• Journal of Microbiology and Biotechnology
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• v.33 no.1
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• pp.51-60
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• 2023

The foodborne illness is the important public health concerns, and the livestock feces are known to be one of the major reservoirs of foodborne pathogens. Also, it was reported that 45.5% of foodborne illness outbreaks have been associated with the animal products contaminated with the livestock feces. In addition, it has been known that the persistence of a pathogens depends on many potential virulent factors including the various virulent genes. Therefore, the first step to understanding the public health risk of livestock feces is to identify and describe microbial communities and potential virulent genes that contribute to bacterial pathogenicity. We used the whole metagenome shotgun sequencing to evaluate the prevalence of foodborne pathogens and to characterize the virulence associated genes in pig and chicken feces. Our data showed that the relative abundance of potential foodborne pathogens, such as Bacillus cereus was higher in chickens than pigs at the species level while the relative abundance of foodborne pathogens including Campylobacter coli was only detected in pigs. Also, the microbial functional characteristics of livestock feces revealed that the gene families related to "Biofilm formation and quorum sensing" were highly enriched in pigs than chicken. Moreover, the variety of gene families associated with "Resistance to antibiotics and toxic compounds" were detected in both animals. These results will help us to prepare the scientific action plans to improve awareness and understanding of the public health risks of livestock feces.

• Journal of Microbiology and Biotechnology
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• v.33 no.1
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• pp.83-95
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• 2023

These days, bacterial detection methods have some limitations in sensitivity, specificity, and multiple detection. To overcome these, novel detection and identification method is necessary to be developed. Recently, NGS panel method has been suggested to screen, detect, and even identify specific foodborne pathogens in one reaction. In this study, new NGS panel primer sets were developed to target 13 specific virulence factor genes from five types of pathogenic Escherichia coli, Listeria monocytogenes, and Salmonella enterica serovar Typhimurium, respectively. Evaluation of the primer sets using singleplex PCR, crosscheck PCR and multiplex PCR revealed high specificity and selectivity without interference of primers or genomic DNAs. Subsequent NGS panel analysis with six artificially contaminated food samples using those primer sets showed that all target genes were multi-detected in one reaction at 10⁸-10⁵ CFU of target strains. However, a few false-positive results were shown at 10⁶-10⁵ CFU. To validate this NGS panel analysis, three sets of qPCR analyses were independently performed with the same contaminated food samples, showing the similar specificity and selectivity for detection and identification. While this NGS panel still has some issues for detection and identification of specific foodborne pathogens, it has much more advantages, especially multiple detection and identification in one reaction, and it could be improved by further optimized NGS panel primer sets and even by application of a new real-time NGS sequencing technology. Therefore, this study suggests the efficiency and usability of NGS panel for rapid determination of origin strain in various foodborne outbreaks in one reaction.

• Journal of Microbiology and Biotechnology
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• v.33 no.2
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• pp.219-227
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• 2023

Lettuce is one of the most consumed vegetables worldwide. However, it has potential risks associated with pathogenic bacterial contamination because it is usually consumed raw. In this study, we investigated the changes in the bacterial community on lettuce (Lactuca sativa L.) in Chungcheong-do, South Korea, and the prevalence of foodborne pathogens on lettuce in different seasons using 16S rRNA gene-based sequencing. Our data revealed that the Shannon diversity index showed the same tendency in term of the number of OTUs, with the index being greatest for summer samples in comparison to other seasons. Moreover, the microbial communities were significantly different between the four seasons. The relative abundance of Actinobacteriota varied according to the season. Family Micrococcaceae was most dominant in all samples except summer, and Rhizobiaceae was predominant in the microbiome of the summer sample. At the genus level, the relative abundance of Bacillus was greatest in spring samples, whereas Pseudomonas was greatest in winter samples. Potential pathogens, such as Staphylococcus and Clostridium, were detected with low relative abundance in all lettuce samples. We also performed metagenome shotgun sequencing analysis on the selected summer and winter samples, which were expected to be contaminated with foodborne pathogens, to support 16S rRNA gene-based sequencing dataset. Moreover, we could detect seasonal biomarkers and microbial association networks of microbiota on lettuce samples. Our results suggest that seasonal characteristics of lettuce microbial communities, which include diverse potential pathogens, can be used as basic data for food safety management to predict and prevent future outbreaks.

• Journal of Microbiology and Biotechnology
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• v.33 no.2
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• pp.188-194
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• 2023

Microbacterium elymi KUDC0405^T was isolated from the rhizosphere of Elymus tsukushiensis from the Dokdo Islands. The KUDC0405^T strain was Gram-stain-positive, non-spore forming, non-motile, and facultatively anaerobic bacteria. Strain KUDC0405^T was a rod-shaped bacterium with size dimensions of 0.3-0.4 × 0.7-0.8 ㎛. Based on 16S rRNA gene sequences, KUDC0405^T was most closely related to Microbacterium bovistercoris NEAU-LLE^T (97.8%) and Microbacterium pseudoresistens CC-5209^T (97.6%). The dDDH (digital DNA-DNA hybridization) values between KUDC0405^T and M. bovistercoris NEAU-LLE^T and M. pseudoresistens CC-5209^T were below 17.3% and 17.5%, respectively. The ANI (average nucleotide identity) values among strains KUDC0405^T, M. bovistercoris NEAU-LLE^T, and M. pseudoresistens CC-5209^T were 86.6% and 80.7%, respectively. The AAI (average amino acid identity) values were 64.66% and 64.97%, respectively, between KUDC0405^T and its closest related type strains. The genome contained 3,596 CDCs, three rRNAs, 46 tRNAs, and three non-coding RNAs (ncRNAs). The genomic DNA GC content was 70.4%. The polar lipids included diphosphatydilglycerol, glycolipid, phosphatydilglycerol, and unknown phospholipid, and the major fatty acids were anteiso-C_17:0 and iso-C_16:0. Strain KUDC0405^T contained MK-12 as the major menaquinone. Based on genotypic, phylogenetic, and phenotypic properties, strain KUDC0405^T should be considered a novel species within the genus Microbacterium, for which we propose the name M. elymi sp. nov., and the type strain as KUDC0405^T (=KCTC 49411^T, =CGMCC1.18472^T).

• Journal of Microbiology and Biotechnology
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• v.33 no.6
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• pp.831-839
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• 2023

Tylosin is a potent veterinary macrolide antibiotic produced by the fermentation of Streptomyces fradiae; however, it is necessary to modify S. fradiae strains to improve tylosin production. In this study, we established a high-throughput, 24-well plate screening method for identifying S. fradiae strains that produce increased yields of tylosin. Additionally, we constructed mutant libraries of S. fradiae via ultraviolet (UV) irradiation and/or sodium nitrite mutagenesis. A primary screening of the libraries in 24-well plates and UV spectrophotometry identified S. fradiae mutants producing increased yields of tylosin. Mutants with tylosin yield 10% higher than the wild-type strain were inoculated into shake flasks, and the tylosin concentrations produced were determined by high-performance liquid chromatography (HPLC). Joint (UV irradiation and sodium nitrite) mutagenesis resulted in higher yields of mutants with enhanced tylosin production. Finally, 10 mutants showing higher tylosin yield were re-screened in shake flasks. The yield of tylosin A by strains UN-C183 (6767.64 ± 82.43 ㎍/ml) and UN-C137 (6889.72 ± 70.25 ㎍/ml) was significantly higher than that of the wild-type strain (6617.99 ± 22.67 ㎍/ml). These mutant strains will form the basis for further strain breeding in tylosin production.

• Journal of Microbiology and Biotechnology
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• v.33 no.5
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• pp.591-599
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• 2023

Fisetin is a bioactive flavonol molecule and has been shown to have antioxidant potential, but its efficacy has not been fully validated. The aim of the present study was to investigate the protective efficacy of fisetin on C2C12 murine myoblastjdusts under hydrogen peroxide (H₂O₂)-induced oxidative damage. The results revealed that fisetin significantly weakened H₂O₂-induced cell viability inhibition and DNA damage while blocking reactive oxygen species (ROS) generation. Fisetin also significantly alleviated cell cycle arrest by H₂O₂ treatment through by reversing the upregulation of p21WAF1/CIP1 expression and the downregulation of cyclin A and B levels. In addition, fisetin significantly blocked apoptosis induced by H₂O₂ through increasing the Bcl-2/Bax ratio and attenuating mitochondrial damage, which was accompanied by inactivation of caspase-3 and suppression of poly(ADP-ribose) polymerase cleavage. Furthermore, fisetin-induced nuclear translocation and phosphorylation of Nrf2 were related to the increased expression and activation of heme oxygenase-1 (HO-1) in H₂O₂-stimulated C2C12 myoblasts. However, the protective efficacy of fisetin on H₂O₂-mediated cytotoxicity, including cell cycle arrest, apoptosis and mitochondrial dysfunction, were greatly offset when HO-1 activity was artificially inhibited. Therefore, our results indicate that fisetin as an Nrf2 activator effectively abrogated oxidative stress-mediated damage in C2C12 myoblasts.

• Journal of Microbiology and Biotechnology
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• v.33 no.4
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• pp.552-558
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• 2023

Levulinic acid (LA) is a valuable chemical used in fuel additives, fragrances, and polymers. In this study, we proposed possible biosynthetic pathways for LA production from lignin and poly(ethylene terephthalate). We also created a genetically encoded biosensor responsive to LA, which can be used for screening and evolving the LA biosynthesis pathway genes, by employing an LvaR transcriptional regulator of Pseudomonas putida KT2440 to express a fluorescent reporter gene. The LvaR regulator senses LA as a cognate ligand. The LA biosensor was first examined in an Escherichia coli strain and was found to be non-functional. When the host of the LA biosensor was switched from E. coli to P. putida KT2440, the LA biosensor showed a linear correlation between fluorescence intensity and LA concentration in the range of 0.156-10 mM LA. In addition, we determined that 0.156 mM LA was the limit of LA detection in P. putida KT2440 harboring an LA-responsive biosensor. The maximal fluorescence increase was 12.3-fold in the presence of 10 mM LA compared to that in the absence of LA. The individual cell responses to LA concentrations reflected the population-averaged responses, which enabled high-throughput screening of enzymes and metabolic pathways involved in LA biosynthesis and sustainable production of LA in engineered microbes.

• Journal of Microbiology and Biotechnology
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• v.33 no.4
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• pp.430-440
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• 2023

The type three secretion system (T3SS) is a major virulence system of Pseudomonas aeruginosa (P. aeruginosa). The effector protein Exotoxin S (ExoS) produced by P. aeruginosa is secreted into the host cells via the T3SS. For the purpose of an experiment on inhibitors with regard to ExoS secretion, we developed a sandwich-type enzyme-linked immunosorbent assay (ELISA) system. Quercetin was selected because it has a prominent ExoS inhibition effect and also is known to have anti-inflammatory and antioxidant effects on mammalian cells. In this study, we investigated the effects of quercetin on the expression and secretion of ExoS using ELISA and Western blot analysis methods. The results showed that the secretion of ExoS was significantly decreased by 10 and 20 µM of quercetin. Also, popB, popD, pscF, and pcrV which are composed of the T3SS needle, are reduced by quercetin at the mRNA level. We also confirmed the inhibitory effect of quercetin on cytokines (IL-6, IL-1β, and IL-18) in P. aeruginosa-infected H292 cells by real-time polymerase chain reaction (PCR) and ELISA. Collectively, quercetin inhibits the secretion of ExoS by reducing both ExoS production and the expression of the needle protein of T3SS. Furthermore, these results suggest that quercetin has the potential to be used as an anti-toxic treatment for the inflammatory disease caused by P. aeruginosa infection.

• Journal of Microbiology and Biotechnology
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• v.33 no.8
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• pp.1023-1029
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• 2023

Biosurfactants reduce surface and interfacial tension due to their amphiphilic properties and are an eco-friendly alternative for chemical surfactants. In this study, a new yeast strain JAF-11 that produces a biosurfactant was selected using drop collapse method, and the properties of the extracts were investigated. The nucleotide sequences of the strain were compared with closely related strains and identified based on the D1/D2 domain of the large subunit ribosomal DNA (LSU) and internal transcribed spacer (ITS) regions. Neodothiora populina CPC 39399^T, the closest species with strain JAF-11, showed a sequence similarity of 97.75% for LSU and 94.27% for ITS, respectively. The result suggests that the strain JAF-11 represents a distinct species that cannot be assigned to any existing genus or species in the family Dothideaceae. Strain JAF-11 produced a biosurfactant reducing the surface tension of water from 72 mN/m to 34.5 mN/m on the sixth day of culture and the result of measuring the critical micelle concentration (CMC) by extracting the crude biosurfactant was found to be 24 mg/l. The molecular weight 502 of the purified biosurfactant was confirmed by measuring the fast atom bombardment mass spectrum. The chemical structure was analyzed by measuring ¹H nuclear magnetic resonance (NMR), ¹³C NMR, and two-dimensional NMRs of the compound. The molecular formula was C₂₆H₄₆O₉, and it was composed of one octanoyl group and two hexanoyl groups to myo-inositol moiety. The new biosurfactant is the first report of a compound produced by a new yeast strain, JAF-11.

• Journal of Microbiology and Biotechnology
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• v.33 no.9
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• pp.1206-1212
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• 2023

The Wnt/β-catenin pathway plays essential roles in regulating various cellular behaviors, including proliferation, survival, and differentiation [1-3]. The intracellular β-catenin level, which is regulated by a proteasomal degradation pathway, is critical to Wnt/β-catenin pathway control [4]. Normally, casein kinase 1 (CK1) and glycogen synthase kinase-3β (GSK-3β), which form a complex with the scaffolding protein Axin and the tumor suppressor protein adenomatous polyposis coli (APC), phosphorylate β-catenin at Ser45, Thr41, Ser37, and Ser33 [5, 6]. Phosphorylated β-catenin is ubiquitinated by the β-transducin repeat-containing protein (β-TrCP), an F-box E3 ubiquitin ligase complex, and ubiquitinated β-catenin is degraded via a proteasome pathway [7, 8]. Colorectal cancer is a significant cause of cancer-related deaths worldwide. Abnormal up-regulation of the Wnt/β-catenin pathway is a major pathological event in intestinal epithelial cells during human colorectal cancer oncogenesis [9]. Genetic mutations in the APC gene are observed in familial adenomatous polyposis coli (FAP) and sporadic colorectal cancers [10]. In addition, mutations in the N-terminal phosphorylation motif of the β-catenin gene were found in patients with colorectal cancer [11]. These mutations cause β-catenin to accumulate in the nucleus, where it forms complexes with transcription factors of the T-cell factor/lymphocyte enhancer factor (TCF/LEF) family to stimulate the expression of β-catenin responsive genes, such as c-Myc and cyclin D1, which leads to colorectal tumorigenesis [12-14]. Therefore, downregulating β-catenin response transcription (CRT) is a potential strategy for preventing and treating colorectal cancer. Plant cytokinins are N⁶-substituted purine derivatives; they promote cell division in plants and regulate developmental pathways. Natural cytokinins are classified as isoprenoid (isopentenyladenine, zeatin, and dihydrozeatin), aromatic (benzyladenine, topolin, and methoxytopolin), or furfural (kinetin and kinetin riboside), depending on their structure [15, 16]. Kinetin riboside was identified in coconut water and is a naturally produced cytokinin that induces apoptosis and exhibits antiproliferative activity in several human cancer cell lines [17]. However, little attention has been paid to kinetin riboside's mode of action. In this study, we show that kinetin riboside exerts its cytotoxic activity against colon cancer cells by suppressing the Wnt/β-catenin pathway and promoting intracellular β-catenin degradation.