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Ryoo, Sungweon,Lee, Jeongsoo,Oh, Jee Youn,Kim, Byeong Ki,Kim, Young,Kim, Je Hyeong,Shin, Chol,Lee, Seung Heon The Korean Academy of Tuberculosis and Respiratory 2018 Tuberculosis and Respiratory Diseases Vol.81 No.3
Background: The number of immigrants with tuberculosis (TB) increases each year in South Korea. Determining the transmission dynamics based on whole genome sequencing (WGS) to cluster the strains has been challenging. Methods: WGS, annotation refinement, and orthology assignment for the GenBank accession number acquisition were performed on two clinical isolates from Chinese immigrants. In addition, the genomes of the two isolates were compared with the genomes of Mycobacterium tuberculosis isolates, from two native Korean and five native Chinese individuals using a phylogenetic topology tree based on the Multiple Alignment of Conserved Genomic Sequence with Rearrangements (Mauve) package. Results: The newly assigned accession numbers for two clinical isolates were CP020381.2 (a Korean-Chinese from Yanbian Province) and CP022014.1 (a Chinese from Shandong Province), respectively. Mauve alignment classified all nine TB isolates into a discriminative collinear set with matched regions. The phylogenetic analysis revealed a rooted phylogenetic tree grouping the nine strains into two lineages: strains from Chinese individuals and strains from Korean individuals. Conclusion: Phylogenetic trees based on the Mauve alignments were supposed to be useful in revealing the dynamics of TB transmission from immigrants in South Korea, which can provide valuable information for scaling up the TB screening policy for immigrants.
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( Sungweon Ryoo ),( Jeongsoo Lee ),( Jee Youn Oh ),( Byeong Ki Kim ),( Young Kim ),( Je Hyeong Kim ),( Chol Shin ),( Seung Heon Lee ) 대한결핵 및 호흡기학회 2018 Tuberculosis and Respiratory Diseases Vol.81 No.3
Background: The number of immigrants with tuberculosis (TB) increases each year in South Korea. Determining the transmission dynamics based on whole genome sequencing (WGS) to cluster the strains has been challenging. Methods: WGS, annotation refinement, and orthology assignment for the GenBank accession number acquisition were performed on two clinical isolates from Chinese immigrants. In addition, the genomes of the two isolates were compared with the genomes of Mycobacterium tuberculosis isolates, from two native Korean and five native Chinese individuals using a phylogenetic topology tree based on the Multiple Alignment of Conserved Genomic Sequence with Rearrangements (Mauve) package. Results: The newly assigned accession numbers for two clinical isolates were CP020381.2 (a Korean-Chinese from Yanbian Province) and CP022014.1 (a Chinese from Shandong Province), respectively. Mauve alignment classified all nine TB isolates into a discriminative collinear set with matched regions. The phylogenetic analysis revealed a rooted phylogenetic tree grouping the nine strains into two lineages: strains from Chinese individuals and strains from Korean individuals. Conclusion: Phylogenetic trees based on the Mauve alignments were supposed to be useful in revealing the dynamics of TB transmission from immigrants in South Korea, which can provide valuable information for scaling up the TB screening policy for immigrants.
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Kang, Heeyoon,Ryoo, Sungweon,Park, Youngkil,Lew, Woojin The Korean Academy of Tuberculosis and Respiratory 2009 Tuberculosis and Respiratory Diseases Vol.67 No.6
Background: Mycobacterial Interspersed Repetitive Units (MIRUs) that are located mainly in intergenic regions dispersed throughout the Mycobacterium tuberculosis genome. The selected MIRU loci, which were composed of a 12-locus set, demonstrated a high power for discrimination of Mycobacterium tuberculosis isolates collected from Kangwon province of Korea. To evaluate its ability to discriminate the M. tuberculosis strains, 45 clinical isolates were genotyped using the methods IS6110 RFLP and MIRU. Methods: All the samples were collected during the period from January 2007 to December 2007 from TB patients, who were residents and registered to a public health center of Kangwon Province in Korea. A total of 45 DNAs were extracted from clinical isolated mycobacterial strains and genotyped using IS6110 RFLP, the MIRU method. Results: We compared the 12-MIRU with IS6110 RFLP in the 45 samples, the 12-locus version offered less discriminatory power (Hunter-Gaston discriminatory index [HGDI]: 0.959 vs 0.998; 57.78% of clustered cases vs 8.89%). Conclusion: This 12-locus MIRU can be useful when additional combinations of other loci for genotyping M. tuberculosis in Korea where the Beijing family strains are dominant.
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( Yoohyun Hwang ),( Sungweon Ryoo ) 대한결핵 및 호흡기학회 2021 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.129 No.-
Background Linezolid (LZD) is an important antibiotic for the treatment of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB), but mutations related to linezolid resistance remain inadequately characterized. In this study we investigated the resistance-related mutations of clinical isolates from MDR and XDR TB patients who failed linezolid treatment, using wholegenome sequencing. Methods We used the Sensititre™ plate to determine the phenotypic drug susceptibility and minimum inhibitory concentration (MIC) of first- and second-line anti-TB drugs, while using resazurin microtiter assay (REMA) for the MIC of linezolid. A total of 25 sequential isolates, previously collected from four MDR/XDR patients who failed LZD treatment, were used in this study. All isolates had whole genome sequencing performed with the Illumina® platform. Results All four TB patients had a form of multidrug-resistant TB, which is defined as a form of TB resistant to at least rifampicin and isoniazid, the first-line anti-TB drugs. Each patients’ isolates collected within three months of beginning LZD treatment were susceptible to LZD, but the isolates from the corresponding patient collected after three months had higher MIC for LZD and developed mutations in LZD resistance linked genes (Table 1). Whole genome sequencing of all strains collected from each patient revealed mutations in the LZD resistance linked genes rplC, rrl, and Rv0678 (Table 2). Conclusions After a given period of time, all four patients acquired resistance to LZD. However, the LZD MIC was below 32ug/ml, a concentration observed in a previous study. The rplC gene affects the ribosomal protein L3, the rrl gene affects the 23S rRNA, and Rv0678 is the regulator for the MmpS5-MmpL5 efflux pump. The combination of mutations in these genes caused the high-level linezolid resistance in each serial clinical isolate.
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( Taeuk Kang ),( Seungkyu Park ),( Sungweon Ryoo ) 대한결핵 및 호흡기학회 2021 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.129 No.-
Background Tuberculosis still remains as intermediate-burden in Korea (Figure 1). For tuberculosis elimination, tuberculosis training/ education play pivotal role. Researches have suggested that provision of appropriate training/education could improve clinical outcome and prevent TB transmission. Despite significant role of TB training/education, TB training/education were poorly investigated. To assess current TB training/education program, identifying current status of TB training/education is inevitable. Hence, in this study, we reviewed articles and programs related to TB training/education in Korea and analyzed. Method A comprehensive review of literature on electronic databases and websites of TB-training providing organizations from year of 1995 to 2021 were undertaken. Result Several organizations from governmental/public sectors such as Korea Disease Control and Prevention Agency (KDCA), Korea National Tuberculosis Association (KNTA), Masan National Tuberculosis Hospital (MNTH), Korea Human Resource Development Institute for Health and Welfare (KOHI), Korea Nurses Association (KNA) were involved in provision of TB training/education. The KDCA conducts activities related to TB control and prevention at national, provincial, and local levels while KNTA, as commissioned by KDCA, provides TB trainings for healthcare workers with collaboration with KNA. In case of MNTH, this medical institute produced 14 TB medical specialists since 1993 and actively engaged in TB education at community level that 67,426 people were educated for TB prevention. Lastly, KOHI institutes provides several TB training/education programs for healthcare workers, medical technologists, and public (Table 1). Conclusion Although sufficient number of TB training/education are being provided. Its quality has never been assessed hence, there is difficulty lies in improvement of TB training/education due to lacking evaluation. Also, due to changing landscape of TB, training/education topics should reflect upcoming demands and be diversified such as inclusion of latent TB, drug-resistant TB and/or HIV/TB. The MNTH, with demonstrated history, is able to meet current demands from TB training/education.
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The Usefulness of Determining the MIC of Newly Developed Anti-TB Drugs with the MBEC Biofilm Assay
( Yoohyun Hwang ),( Dagyum Lee ),( Sungweon Ryoo ) 대한결핵 및 호흡기학회 2021 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.129 No.-
Background Drug-resistance is one of the ongoing topics in the treatment of bacterial infections; in the case of tuberculosis (TB), effective treatment is inhibited by the presence of the atypical mycobacterial cell wall and biofilm, which prevents antibiotic penetration into the cell. Additionally, the biofilm layer increases the mycobacterial antibiotic tolerance and delays the host immune response, which encourages drug-resistance. Methods We used the MBEC Assay Biofilm Inoculator (Innovotech) with Mycobacterium abscessus (M. abs) in this study. 96 pegs set on the lid of the assay was placed on a 96 well plate with an OD600 0.01 of M. abs, then incubated at rpm 80 for 3 days at 37℃ to form biofilm on the pegs. The pegs were then transferred to a new 96 well plate with various drug concentrations and incubated again under same conditions. Then 0.25% resazurin was added to measure minimum inhibitory concentration (MIC). Results Several antibiotics effective when used against the cells of M. abs in the resazurin microtiter assay (REMA) were ineffective in the treatment of M. abs cells with biofilm. In the presence of biofilm, the MIC50 for antibiotics were up to almost 3 times the MIC50 concentrations of aerobic M. abs growth conditions without biofilm. Conclusions Biofilm formation of mycobacteria is one of the complications behind the treatment of mycobacterium infections, including Mycobacterium tuberculosis, one of the leading infectious respiratory diseases. The presence of biofilm skews Results of MIC researched in vitro to the point of ineffectiveness in vivo. More prolific use of the MBEC biofilm assay to determine the effectiveness of newly developed drugs are needed before introducing them to most in vivo experiments.
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( Da-gyum Lee ),( Sungweon Ryoo ),( Yoohyun Hwang ),( Eun-jin Park ),( Jung-hyun Kim ) 대한결핵 및 호흡기학회 2021 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.129 No.-
Background Mycobacterium abscessus (M. abscessus) causes chronic pulmonary infections and is the most difficult Nontuberculous Mycobacteria (NTM) to treat due to its resistance to current antimicrobial drugs, with a treatment success rate of 45.6 %. Thus, novel treatment drugs are needed, of which we identified the drug Clomiphene Citrate (CC), which treats infertility in women, to exhibit inhibitory activity against M. abscessus. Methods To assess the potential of CC as a treatment for M. abscessus pulmonary diseases, we measured its efficacy in vitro and established the intracellular activity of CC against M. abscessus in human macrophages. Results CC significantly inhibited the growth of not only wild-type M. abscessus strains, but also clinical isolate strains and clarithromycin (CLR)-resistant strains of M. abscessus. CC’s drug-efficacy did not have a significant cytotoxicity in the infected macrophages. Furthermore, CC worked in anaerobic non-replicating conditions as well as in the presence of biofilm. Conclusions The Results of this in vitro study on M. abscessus activity suggest that CC is a potential new drug for the treatment of M. abscessus infections.
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Antiviral Effect of Low Molecular Weight Chitooligosaccharide Against SARS-CoV-2 in Vitro
( Donghwan Jang ),( Dagyum Lee ),( Jihee Jung ),( Sungweon Ryoo ) 대한결핵 및 호흡기학회 2021 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.129 No.-
Background We tried to assure the natural polysaccharide would be used as a disinfectant for Coronavirus disease 2019. Previous studies already reported that the chitosans are effective substances for viral diseases. Here, we showed that three different molecular weight chitosan-based substances (chitosan, chitooligosaccharide, and water soluble chitosan) have direct antiviral properties against SARS-CoV-2 in vitro using Vero cells. Methods The cytotoxicity of chitosan-based substances was measured by MTT assay. The antiviral effect was confirmed by quantitative viral RNA targeting the RdRp gene and plaque assay. The reduction of nucleocapsid protein expression in the virus-infected cells was confirmed by fluorescence microscopy. Results The chitosan-based substances have low cytotoxicity to Vero cells. Among the chitosan-based substances tested, chitooligosaccharide decreased the expression of SARS-CoV-2 nucleocapsid protein of SARS-CoV-2 infected cells and reduced SARS-CoV-2 virus infection in a concentration-dependent manner. Conclusions In conclusion, low molecular weight chitooligosaccharide can serve as an antiviral natural disinfectant for SARS-CoV-2 in vitro.
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Testing the Efficacy of COVID-19 Vaccine and Therapeutic Candidates Using Vero 76 Cell Line
( Noori Lee ),( Donghwan Jang ),( Munki Yoo ),( Sungweon Ryoo ) 대한결핵 및 호흡기학회 2021 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.129 No.-
Background COVID-19 is a respiratory infectious disease that first emerged in Wuhan, China in December 2019 and has spread worldwide. It is necessary to develop preventive vaccines and therapeutics for overcoming COVID-19. For this purpose, , in vitro efficacy testing of the applicants is essential in the early stages of development. We want to share our experience of in vitro efficacy testing Methods using SARS-CoV-2 through this presentation. Methods All tests using SARS-CoV-2 were performed within the Biosafely Level 3 (BL3) facility of Masan National Tuberculosis Hospital (MNTH). And the protocols were approved by the Institutional Biosafety Committee (IBC) of MNTH prior to the study. Vaccine candidate neutralizing Serums were obtained by collecting blood from animals immunized with vaccine candidates. In 6 well plates, the Vero 76 cells were pre-culture to sub-confluence. The serum dilution and the virus was mixed in a 1:1 volume and neutralized. The serumvirus mixture was added to the cells for infection and the uninfected virus was removed. Add media containing 1% agarose, and solidify completely. After culturing for 3 days, fix with formaldehyde, remove agarose, and stain with crystal violet. The neutralizing effect was calculated by counting the plaques and comparing them with the control. Therapeutic candidates Infect SARS-CoV-2 to Vero 76 cells with an MOI 0.01. Add the test substance prepared for each concentration and Remdesivir, a positive control, and incubate. The culture medium and cell lysate are collected, the therapeutic effect is confirmed by comparing Results with the control group by Methods such as plaque assay, qPCR and WB. Results We established our optimized in vitro protocol for testing the efficacy of vaccines and therapeutic candidates against COVID-19 quantitatively. Conclusion The established Methods would be useful in confirming the efficacy of COVID-19 vaccine candidates and therapeutic candidates in the preclinical stage.
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