• Journal of Microbiology and Biotechnology
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• v.30 no.9
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• pp.1335-1342
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• 2020

The identification of bacterial pathogens to humans is critical for environmental microbial risk assessment. However, current methods for identifying pathogens in environmental samples are limited in their ability to detect highly diverse bacterial communities and accurately differentiate pathogens from commensal bacteria. In the present study, we suggest an improved approach using a combination of identification results obtained from multiple databases, including the multilocus sequence typing (MLST) database, virulence factor database (VFDB), and pathosystems resource integration center (PATRIC) databases to resolve current challenges. By integrating the identification results from multiple databases, potential bacterial pathogens in metagenomes were identified and classified into eight different groups. Based on the distribution of genes in each group, we proposed an equation to calculate the metagenomic pathogen identification index (MPII) of each metagenome based on the weighted abundance of identified sequences in each database. We found that the accuracy of pathogen identification was improved by using combinations of multiple databases compared to that of individual databases. When the approach was applied to environmental metagenomes, metagenomes associated with activated sludge were estimated with higher MPII than other environments (i.e., drinking water, ocean water, ocean sediment, and freshwater sediment). The calculated MPII values were statistically distinguishable among different environments (p < 0.05). These results demonstrate that the suggested approach allows more for more accurate identification of the pathogens associated with metagenomes.

• The Plant Pathology Journal
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• v.26 no.4
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• pp.313-320
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• 2010

Genetic instability of the rice blast fungus Magnaporthe oryzae has been suggested as a major factor underlying the rapid breakdown of host resistance in the field. However, little information is available on the mechanism of genetic instability. In this study, we assessed the stability of repetitive DNA elements and several key phenotypic traits important for pathogenesis after serially transferring two isolates though rice plants and an artificial medium. Using isolate 70-15, we obtained a total of 176 single-spore isolates from 10 successive rounds of culturing on artificial medium. Another 20 isolates were obtained from germ tubes formed at the basal and apical cells of 10 three-celled conidia. Additionally, 60 isolates were obtained from isolate KJ201 after serial transfers through rice plants and an artificial medium. No apparent differences in phenotypes, including mycelial growth, conidial morphologies, conidiation, conidial germination, appressorium formation, and virulence, or in DNA fingerprints using MGR586, MAGGY, Pot2, LINE, MG-SINE and PWL2 as probes were observed among isolates from the same parent isolate. Southern hybridization and sequence analysis of two avirulence genes, AVR-Pita1 and AVR-Pikm, showed that both genes were also maintained stably during 10 successive generations on medium and plants. However, one reversible loss of restriction fragments was found in the telomere-linked helicase gene (TLH1) family, suggesting some telomere regions may be more unstable than the rest of the genome. Taken together, our results suggest that phenotype and genotype of M. oryzae isolates do not noticeably change, at least up to 10 successive generations on a cultural medium and in host plants.

• Journal of Life Science
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• v.32 no.1
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• pp.56-62
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• 2022

This study was investigated to evaluate the potential of Stewartia koreana Nakai as an oral healthcare material. The inhibitory effects of extracts on the biofilm formation and fimbriae genes expression of Porphyromonas gingivalis were determined by minimal inhibitory concentrations (MIC), biofilm biomass staining, SEM, and qRT-PCR analysis. The S. koreana Nakai branch was extracted into 70% ethanol, and bacteriostatic MIC of extracts against P. gingivalis were 0.6 mg/ml. In P. gingivalis cultures treated with 0.2-2.0 mg/ml of extract, biofilm production rate was significantly decreased in a concentration-dependent manner. The morphology of treated and untreated samples was observed by SEM, and cell aggregation and biofilm were only observed in those treated with extract. Subsequently, qRT-PCR analysis showed that the mRNA expression on fimbriae genes fimA and fimB was suppressed in a concentration-dependent manner. Based on these results, it can be suggested that S. koreana branch extract has the potential to be used as naturally derived oral healthcare material because of its bacteriostatic action and inhibition of P. gingivalis biofilm formation.

• 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.

• Korean Journal of Microbiology
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• v.44 no.2
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• pp.85-92
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• 2008

Pseudomonas aeruginosa is an opportunistic human pathogen, causing a wide variety of infections including cystic fibrosis, microbial keratitis, and burn wound infections. The cell-to-cell signaling mechanism known as quorum sensing (QS) plays a key role in these infections and the QS systems of P. aeruginosa have been most intensively studied. While many literatures that introduce the QS systems of P. aeruginosa have mostly focused on two major acyl-homo serine lactone (acyl-HSL) QS signals, N-3-oxododecanoyl homoserine lactone (3OC12) and N-butanoyl homoserine lactone (C4), several new signal molecules have been discovered and suggested for their significant roles in signaling and virulence of P. aeruginosa. One of them is PQS (Pseudomonas quinolone signal; 2-heptyl-3-hydroxy-4-quinolone), which is now considered as a well-characterized major signal meolecule of P. aeruginosa. In addition, recent researches have also suggested some more putative signal molecules of P. aeruginosa, which are diketopiperazines (DKPs) and pyocyanin. DKPs are cyclic dipeptides and structurally diverse depending on what amino acids are involved in composition. Some DKPs from the culture supernatant of P. aeruginosa are suggested as new diffusible signal molecules, based on their ability to activate Vibrio fischeri LuxR biosensors that are previously considered specific for acyl-HSLs. Pyocyanin (1-hydroxy-5-methyl-phenazine), one of phenazine derivatives produced by P. aeruginosa is a characteristic blue-green pigment and redox-active compound. This has been recently suggested as a terminal signaling factor to upregulate some QS-controlled genes during stationary phase under the mediation of a transcription factor, SoxR. Here, details about these newly emerging signaling molecules of P. aeruginosa are discussed.

• Journal of Food Hygiene and Safety
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• v.38 no.6
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• pp.544-550
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• 2023

To develop a functional probiotic that inhibits gingipain, a major virulence factor of Porphyromonas gingivlais (P. gingivalis), we screened over 30 probiotic strains for their ability to inhibit gingipian activity. We investigated the inhibition of expression of gingipain genes kgp, rgpA, and rgpB as well as gingipain activity, using freeze dried cell-free supernatants of Weisiella cibaria SPM402 (WC402) and Lactobacillus paracasei SMP412 (LP412), both of which demonstrated antibacterial activity against P. gingivalis. Thus, it was verified that kgp expression was reduced by approximately 0.71±0.02 folds and rgpB expression was reduced by approximately 0.71±0.14 folds at a concentration of WC402 10 mg/mL. Meanwhile, at the same concentration of 10 mg/mL of LP412, kgp expression was reduced by approximately 0.19±0.08 folds, rgpA expression was reduced by approximately 0.09±0.02 folds, and rgpB expression was reduced by approximately 0.24±0.03 folds. At a concentration of 10 mg/mL, Kgp activity was inhibited by approximately 78.65±3.58% (cell associated gingipain, CAG), 82.45±1.22% (cell-free gingipain, CFG) by WC402 and 80.71±2.11% (CAG), and 85.81±0.05% (CFG) by LP412 respectively. Rgp activity was also effectively inhibited by approximately 78.6±1.01% (CAG), 86.78±0.47% (CFG) and 82.93±1.26% (CAG), 88.81±0.36% (CFG) by WC402 and LP412 respectively. Based on these results, W. cibaria SPM402 and L. paracasei SPM412 can be regarded as functional probiotics with the ability to inhibit gingipain activity and exhibit antibacterial effects against P. gingivalis.

• Korean Journal of Clinical Laboratory Science
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• v.50 no.2
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• pp.100-109
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• 2018

The emergence and dissemination of multidrug-resistant (MDR) Acinetobacter baumannii isolates have been reported worldwide, with most of these possessing the ability to form biofilms. Biofilm formation is an important virulence factor associated with the resistance to disinfection and desiccation. This study examined the genetic basis of antimicrobial resistance mechanisms of biofilm-forming A. baumannii clinical isolates. Imaging and quantification of biofilms were performed by a crystal violet assay and 46 biofilm-forming A. baumannii isolates were selected. Subsequently, 16 isolates belonging to different clones were identified using REP-PCR, and detection of the antimicrobial determinants in the isolates was carried out. The 16 isolates included 9 non-MDR and 7 MDR isolates. The mean biomass $OD_{560}$ values of the non-MDR (0.96) and MDR (1.05) isolates differed but this difference was not significant. In this study, most biofilm-forming MDR A. baumannii isolates contained various antimicrobial resistance determinants ($bla_{OXA-23}$, armA, and mutations of gyrA and parC). On the other hand, most biofilm-forming non-MDR A. baumannii isolates did not contain antimicrobial resistance determinants. These results suggest that there is little correlation between the biofilm-forming ability and antimicrobial susceptibility in A. baumannii isolates. In addition, the emergence of MDR A. baumannii clinical isolates is generally caused by mutations of the genes associated with antimicrobial resistance and/or the acquisition of various antimicrobial resistance determinants.

• Journal of Life Science
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• v.17 no.3 s.83
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• pp.386-395
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• 2007

A total of 27 strains of Vibrio parahaemolyticus (18 strains isolated from Korea and 9 strains from Japan) were serotyped and examined for biochemical characteristics, antimicrobial susceptibility patterns, cytotoxicity assay, thermostable direct hemolysin (TDH) production and molecular epidemiology. Using polymerase chain reaction (PCR) method and DNA probe hybridization method, the strains were tested for toxR, tdh, trh and ORF 8 genes. The V. parahaemolyticus isolated from patients were belonged to 8 different serotypes : O3:K6, O1:K38, O3:K57, O4:K9, O4:Kl2, O4:K68, O5:Kl5 and O6:K46. Urease-positive strain possessed the trh gene, and conversely, urease-negative strains lacked the gene, indicating that urease production by V. parahemolyticus strains strongly correlates with the possession of the trh gene. Most strains showed multiple resistant to more than three antibiotics and the antibiogram could be classified into 6 group (I to VI). All of the O3:K6 strains isolated in South Korea and Japan producted TDH at high levels. The TDH titers ranged between 256 and 2.048, and the average titer was 1009. To distinguish the new and increasingly common V. parahaemolyticus strains from clinical isolates, ORF 8 is a useful genetic marker. After Southern hybridization, the HindIII restriction fragment patterns of the tdh gene were grouped one type, respectively. One type showed two bands one of which was 4.3kb and the other was 11.5kb in size. Variation between the O3:K6 serotype are minor when compared to the differences seen with the non O3:K6 strains. The migration patterns of Not I -digested of the total DNA of the O3:K6 strains were similar, and only slight variations were observed between the serotypes. By contrast, the O3:K6 strains and non O3:K6 had markedly different profiles. In conclusion, Random amplified polymorphic DNA (RAPD) profile using appropriate primers was an effective epidemiological marker.

• Korean Journal of Cognitive Science
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• v.11 no.2
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• pp.37-51
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• 2000

The present study compared computer users target-selection response patterns when the targets were varied in terms of their relative location and distance from the current position of the cursor. In Experiment 1, where the mouse was used as an input device, the effects of different directions and distances of simple target(small rectangle) on target-selection response were investigated. The results of Experiment 1 can be summarized as follows: (1) Overshooting was more frequent than either undershooting or correct movement and (2) this tendency was more prominent when the targets were presented in the oblique direction or in farther location from the current cursor position. (3) Although the overshooting and undershooting were more frequent in the oblique direction, the degree of deviation was larger in horizontal and vertical direction. (4) Time spent in moving the mouse rather than that spent in planning, calibrating or clicking was found to be the most critical factor in determining total response time. In Experiment 2, effects of the font size and line-height of the target on target-selection response were compared with regard to two types of input devices(keyboard vs. mouse). The results are as follows: (1) Mouse generally yielded shorter target-selection time than keyboard. but this tendency was reversed when the targets were presented in horizontal and vertical directions. (2) In general, target-selection time was the longest in the condition of font size of 10 and line-height of 100%, and the shortest in the condition of font size of 12 and line-height of 150%. (3) When keyboard was used as the input device, target-selection time was shortest in the 150% line-height condition, whereas in the mouse condition, target-selection time tended to be increased as the line-height increased. which resulted in the significant interaction effect between input device and line-height. Finally, several issues relating to human-computer interaction were discussed based on the results of the present study.

• Korean Journal of Clinical Laboratory Science
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• v.48 no.2
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• pp.82-87
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• 2016

Enterotoxigenic Bacteroides fragilis (ETBF) produces enterotoxins known to be a virulence factor. Three isotypes of the B. fragilis toxin (BFT) gene have been identified: bft-1, bft-2, and bft-3. We investigated the presence of bft isotypes in clinical B. fragilis isolates and the antimicrobial resistance of BFT-negative and BFT-positive isolates. Overall, 537 B. fragilis isolates were collected from extraintestinal specimens over 8 years (2006~2013) from a university hospital in Korea. Samples were analyzed by multiplex PCR to identify the bft gene isotypes. Additionally, the antimicrobial susceptibility of 107 B. fragilis isolates (74 BFT-negative and 33 BFT-positive) was examined by the CLSI agar dilution method. PCR revealed a total bft gene detection rate of 30%, while 33% and 29% of blood and other extraintestinal isolates contained the gene, respectively. Among ETBF isolates, the most common isotype was bft-1 gene, followed by bft-2 and bft-3 (bft-1 77%, bft-2 14%, bft-3 9%). Resistance rates (%) for BFT-negative and positive isolates differed in response to various antimicrobial agents, with 3%, 5%, 1% and 38% of BFT-negative isolates and 3%, 6%, 3% an 42% of BFT-positive isolates being resistant to piperacillin-tazobactam, cefoxitin, imipenem, and clindamycin, respectively. Interestingly, neither BFT-negative nor positive isolates showed antimicrobial resistance to chloramphenicol and metronidazole. Overall, the proportion of ETBF from blood was similar to that of other extraintestinal sites and the bft-1 gene was the predominant isotype. Higher antimicrobial resistance rates were found in BFT-positive isolates than BFT-negative isolates, but these differences were not statistically significant.