• Tuberculosis and Respiratory Diseases
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• v.80 no.3
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• pp.270-276
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• 2017

Background: A disputed rpoB mutation is a specific type of rpoB mutation that can cause low-level resistances to rifampin (RIF). Here, we aimed to assess the frequency and types of disputed rpoB mutations in Mycobacterium tuberculosis isolates from South Korea. Methods: Between August 2009 and December 2015, 130 patients exhibited RIF resistance on the MTBDRplus assay at Asan Medical Center. Among these cases, we identified the strains with disputed rpoB mutation by rpoB sequencing analysis, as well as among the M. tuberculosis strains from the International Tuberculosis Research Center (ITRC). Results: Among our cases, disputed rpoB mutations led to RIF resistance in at least 6.9% (9/130) of the strains that also exhibited RIF resistance on the MTBDRplus assay. Moreover, at the ITRC, sequencing of the rpoB gene of 170 strains with the rpoB mutation indicated that 23 strains (13.5%) had the disputed mutations. By combining the findings from the 32 strains from our center and the ITRC, we identified the type of disputed rpoB mutation as follows: CTG511CCG (L511P, n=8), GAC516TAC (D516Y, n=8), CTG533CCG (L533P, n=8), CAC526CTC (H526L, n=4), CAC526AAC (H526N, n=3), and ATG515GTG (M515V, n=1). Conclusion: Disputed rpoB mutations do not seem to be rare among the strains exhibiting RIF resistance in South Korea.

• Tuberculosis and Respiratory Diseases
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• v.50 no.3
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• pp.334-342
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• 2001

Background : RpoB gene mutations have been found in about 96-98% of rifampicin (RMP)-resistant Mycobacterium tuberculosis. Recent reports confirm that the in laboratory settings a rpoB gene mutation can be used as a surrogate marker for multi-drug resistant tuberculosis. However, its usefulness in clinical applications has not been evaluated. This study was performed to confirm whether mutation analysis of the rpoB gene of M. tuberculosis is useful in clinical settings. Methods : The medical records of 33 patients in whom rpoB gene analysis was conducted using an INNOLiPA Rif. TB assay (LiPA) from June, 1998, to July, 2000, at the Asan Medical Center were retrospectively reviewed in 33 patients. The clinical characteristics in addition to the drug susceptibility and LiPA results were analyzed. The drug susceptibility test was considered as a gold standard method for M. tuberculosis susceptibility and these results were compared with those of the rpoB gene study and sequencing analysis. Sequencing analysis of the rpoB gene was done in cases where there was a discrepancy between the results of the drug susceptibility an d rpoB gene study. Results : The mean age and sex ratio was $42{\pm}18$, and 24:9 (M:F), respectively. There were 19 RMP susceptible (58%) and 14 RMP-resistant cases (42%) according to the rpoB gene study. The mean time from the request to reporting the results of the rpoB gene study was $5.2{\pm}2.6$ days. The mean gap from reporting the rpoB gene study to reporting the susceptibility was $56{\pm}35$ days. Twenty-eight cases (85%) showed identical results compared with the drug susceptibility results, whereas five cases (15%) showed contradictory results. When compared with the sequencing analysis, of the five cases that showed contradictory results, two had LiP A analysis errors and the remaining three were identical to the sequencing results. The rpoB gene study was of assistance in choosing the appropriate drugs in 28 cases (85%). Conclusions : An rpoB gene study using an LiP A assay was useful in rapidly diagnosing RMP-resistant tuberculosis, which enabled a proper choice of the appropriate drugs in clinical practices. However, an LiPA assay always should be performed in conjunction with microscopy, culture, and susceptibility tests.

• Journal of Microbiology and Biotechnology
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• v.27 no.6
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• pp.1128-1137
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• 2017

The aim of this study was to explore the accuracy and feasibility of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in identifying bacteria from environmental sources, as compared with rpoA gene sequencing, and to evaluate the occurrence of bacteria of the genus Enterococcus in wild birds. In addition, a phyloproteomic analysis of certain Enterococcus species with spectral relationships was performed. The enterococci were isolated from 25 species of wild birds in central Europe (Poland). Proteomic (MALDI-TOF MS) and genomic (rpoA gene sequencing) methods were used to identify all the isolates. Using MALDI-TOF MS, all 54 (100%) isolates were identified as Enterococcus spp. Among these, 51 (94.4%) isolates were identified to the species level (log(score) ${\geq}2.0$), and three isolates (5.6%) were identified at a level of probable genus identification (log(score) 1.88-1.927). Phylogenetic analysis based on rpoA sequences confirmed that all enterococci had been correctly identified. Enterococcus faecalis was the most prevalent enterococcal species (50%) and Enterococcus faecium (33.3%) the second most frequent species, followed by Enterococcus hirae (9.3%), Enterococcus durans (3.7%), and Enterococcus casseliflavus (3.7%). The phyloproteomic analysis of the spectral profiles of the isolates showed that MALDI-TOF MS is able to differentiate among similar species of the genus Enterococcus.

• Tuberculosis and Respiratory Diseases
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• v.83 no.4
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• pp.289-294
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• 2020

Background: Line probe assay (LPA) is standard diagnostic tool to detect multidrug resistant tuberculosis. Non-interpretable (NI) results in LPA (complete missing or light wild-type 3 and 8 bands with no mutation band in rpoB gene region) poses a diagnostic challenge. Methods: Sputum samples obtained between October 2016 and July 2017 at the Intermediate Reference Laboratory, All India Institute of Medical Sciences Hospital, New Delhi, India were screened. Smear-positive and smear-negative culture-positive specimens were subjected to LPA Genotype MTBDRplus Ver 2.0. Smear-negative with culture-negative and culture contamination were excluded. LPA NI samples were subjected to phenotypic drug susceptibility testing (pDST) using MGIT-960 and sequencing. Results: A total of 1,614 sputum specimens were screened and 1,340 were included for the study (smear-positive [n=1,188] and smear-negative culture-positive [n=152]). LPA demonstrated 1,306 (97.5%) valid results with TUB (Mycobacterium tuberculosis) band, 24 (1.8%) NI, three (0.2%) valid results without TUB band, and seven (0.5%) invalid results. Among the NI results, 22 isolates (91.7%) were found to be rifampicin (RIF) resistant and two (8.3%) were RIF sensitive in the pDST. Sequencing revealed that rpoB mutations were noted in all 22 cases with RIF resistance, whereas the remaining two cases had wild-type strains. Of the 22 cases with rpoB mutations, the most frequent mutation was S531W (n=10, 45.5%), followed by S531F (n=6, 27.2%), L530P (n=2, 9.1%), A532V (n=2, 9.1%), and L533P (n=2, 9.1%). Conclusion: The present study showed that the results of the Genotype MTBDRplus assay were NI in a small proportion of isolates. pDST and rpoB sequencing were useful in elucidating the cause and clinical meaning of the NI results.

• Tuberculosis and Respiratory Diseases
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• v.43 no.6
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• pp.842-851
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• 1996

Background : Rifampicin(RFP) is a key component of the antituberculous shon-course chemotherapy and the RFP-resistance is a marker of multi-drug resistant(MDR) M. tuberculosis. rpoB gene encodes the ${\beta}$-subunit of RNA polymerase of M. tuberculosis which is the target of RFP. Recent reports show that rpoB gene mutations are the cause of RFP resistance of M. tuberculosis and the main mechanism of rpoB gene mutation is point mutation. And PCR-SSCP is a rapid and easy method for detecting point mutations. So we performed PCR-SSCP of rpoB gene of M. tuberculosis and compared the result with traditional RFP sensitivity test. Method : The 27 RFP sensitive M. tuberculosis culture isolates and 25 RFP resistant isolates were evaluated. The RFP sensitivity test was done at the Korean Tuberculosis istitute. The DNA was extracted by bead beater method and was amplified with primers TR-8 and TR-9 in a 20ul PCR reaction containing 0.1ul(luCi) [${\alpha}-^{32}P$] - dCTP. After amplification, SSCP was done using non-denaturaring polyacrylamide gel electrophoresis. Then direct sequencing was done in cases of different eletrophoretic mobility compared with that of H37Rv. In 19 cases, we compared PCR-SSCP results with patient's clinical course and the results of traditional RFP sensitivity test. Results : 1) All 27 RFP sensitive M. tuberculosis isolates showed the same electrophoretic mobility compared with that of H37Rv. And all 25 RFP resistant M. tuberculosis isolates showed different electrophoretic mobility. 2) The mechanism of rpoB gene mutation of M. tuberculosis is mainly point mutation. 3) The PCR-SSCP results correlate well with traditional RFP sensitivity and patient's clinical response to antituberculous treatment. Conclusion: The PCR-SSCP of rpoB gene is a very sensitive and rapid mehod in detecting RFP- resistant M. tuberculosis.

• Biomedical Science Letters
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• v.25 no.1
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• pp.40-53
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• 2019

Of the Acinetobacter spp., A. baumannii (genospecies 2) is the most clinically significant in terms of hospital-acquired infections worldwide. It is difficult to perform Acinetobacter-related taxonomy using phenotypic characteristics and routine laboratory methods owing to clusters of closely related species. The ability to accurately identify Acinetobacter spp. is clinically important because antimicrobial susceptibility and clinical relevance differs significantly among the different genospecies. Based on the medical importance of pathogenic Acinetobacter spp., the distribution and characterization of Acinetobacter spp. isolates from 123 clinical samples was determined in the current study using four typically applied bacterial identification methods; partial rpoB gene sequencing, amplified rRNA gene restriction analysis (ARDRA) of the intergenic transcribed spacer (ITS) region of the 16~23S rRNA, the $VITEK^{(R)}$ 2 system (an automated microbial identification system) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). A. baumannii isolates (74.8%, 92/123) were the most common species, A. nosocomialis (10.6%, 13/123) and A. pittii isolates (7.5%, 9/123) were second and third most common strains of the A. calcoaceticus-A. baumannii (ACB) complex, respectively. A. soli (5.0%, 6/123) was the most common species of the non-ACB complex. RpoB gene sequencing and ARDRA of the ITS region were demonstrated to lead to more accurate species identification than the other methods of analysis used in this study. These results suggest that the use of rpoB genotyping and ARDRA of the ITS region is useful for the species-level identification of Acinetobacter isolates.

• Journal of the Korean Wood Science and Technology
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• v.46 no.4
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• pp.402-414
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• 2018

Species identification of wood provides important information for archaeology, restoration of cultural assets, preventing illegal logging, and more. Wood species are usually identified based on their anatomical features with the use of a microscope. However, this method may not be able to distinguish between anatomically similar species or subspecies. To overcome this problem, wood species need to be identified at the molecular level using DNA sequencing. However, unlike living plant cells, wood is difficult to pulverize using a mortar, and DNA extraction from dried wood is challenging. To solve these problems, we propose a pretreatment method in which wood is pulverized using 60-grit sandpaper and hydrated with water for 2 days. Using this method, we were able to stably amplify the rpoB gene from the extracted DNA of Pinus rigida. In addition, sequence analysis of the rpoB gene revealed six single nucleotide polymorphisms (SNPs), which classified the rpoB sequences in the genus Pinus into five groups. Our data indicate that although these SNPs were not suitable for species identification, they can potentially be used to determine the origin of different wood subspecies or individual samples of wood.

• Journal of Microbiology and Biotechnology
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• v.11 no.2
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• pp.287-293
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• 2001

A fast and easy PCR-SSCP method was developed and assessed for the early detection of rifampin-resistant Mycobacterium leprae in skin biopsy samples from Korean leprosy patients. The 190 bp of the rpoB gene, in which mutation is known to cause resistance to rifampin, was amplified by PCR and then analyzed by SSCP and DNA sequencing, All PCR products showing mobility shift on PCR-SSCP contained mutations, demonstrating that this method can be used for an early diagnositic method to detect a putative rifampin-resistant M. leprae strain. DNA sequence analysis revealed that 19 of 34 patient samples contained M. leprae strains with missense mutations in the rpoB gene: five were the same mutations previously reported to cause rifampin resistance and eight were the new type of mutatios that likely cause rifampin resistance. These newly identified dmutations, whose all five cytosine bases of four amino acids were substitued with thymine, were found at different sites from those reported in Mycobacterium tuberculosis or M. leprae. Therefore, they may provide additional clues to understand the molecular biological basis on the rifampin resistance of M. leprae.

• Tuberculosis and Respiratory Diseases
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• v.49 no.5
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• pp.546-557
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• 2000

Background : Oligonucleotide chip technology has proven to be a very useful tool in the rapid diagnosis of infectious disease. Rifampin resistance is considered as a useful marker of multidrug-resistance in tuberculosis. Mutations in the rpoB gene coding $\beta$ subunit of RNA polymerase represent the main mechanism of rifampin resistance. The purpose of this study was to develop a diagnosis kit using oligonucleotide chip for the rapid and accurate detection of rifampin-resistance in Mycobacterium tuberculosis. Method : The sequence specific probes for mutations in the rpoB gene were designed and spotted onto the glass slide, oligonucleotide chip. 38 clinical isolates of Mycobacterium were tested. A part of rpoB was amplified, labelled, and hybridized on the oligonucleotide chip with probes. Results were analyzed with a laser scanner. Direct sequencing was done to verify the results. Result : The low-density oligonucleotide chip design어 to determine the specific mutations in the rpoB gene of M. tuberculosis accurately detected rifampin resistance associated with mutations in 28 clinical isolates. Mutations at codons 531, 526, and 513 were confirmed by direct sequencing analysis. Conclusion : Mutant detection using oligonucleotide chip technology is a reliable and useful diagnostic tool for the detection of multidrug-resistance in M. tuberculosis.

• Microbiology and Biotechnology Letters
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• v.50 no.2
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• pp.301-309
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• 2022

Helicobacter pylori (H. pylori) establishes infection in the human gastric mucosa for a long time and causes severe gastric diseases such as peptic ulcer and gastric cancer. When H. pylori is exposed to the antibacterial agents or inhibitors, the expression of pathogenic associated genes could be altered. In this study, we analyzed the transcriptional changes of H. pylori genes induced by zerumbone treatment. RNA expression changes were analyzed using next-generation sequencing (NGS), and then reverse transcription-polymerase chain reaction (RT-PCR) was performed to verify the results. As a result of NGS analysis, a total of 23 out of 1,632 genes were differentially expressed by zerumbone treatment. RT-PCR confirmed that zerumbone treatment regulated the expression level of 14 genes. Among the genes associated with DNA replication, transcription, virulence factors and T4SS components, 10 genes (dnaE, dnaQ, rpoA, rpoD, secA, flgE, flhA, virB5, virB8 and virB9) were significantly down-regulated and 4 genes (flaA, flaB, virB4 and virD4) were up-regulated. The results of our current study imply that zerumbone might be a potential therapeutic agent for H. pylori infection by regulating factors related to various H. pylori pathogenicity.