• Tuberculosis and Respiratory Diseases
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• 제80권2호
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• pp.159-168
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• 2017

Background: Streptomycin (SM) is recommended by the World Health Organization (WHO) as a part of standard regimens for retreating multidrug-resistant tuberculosis (MDR-TB) cases. The incidence of MDR-TB in retreatment cases was 19% in Thailand. To date, information on SM resistance (SMR) gene mutations correlated to the SMR of Mycobacterium tuberculosis Thai isolates is limited. In this study, the mutations in rpsL, rrs, gidB, and whiB7 were investigated and their association to SMR and the lineage of M. tuberculosis were explored. Methods: The lineages of 287 M. tuberculosis collected from 2007 to 2011 were identified by spoligotyping. Drug susceptibility profiles were evaluated by the absolute concentration method. Mutations in SMR genes of 46 SM-resistant and 55 SM-susceptible isolates were examined by DNA sequencing. Results: Three rpsL (Lys43Arg, Lys88Arg, and Lys88Thr) and two gidB (Trp45Ter and Gly69Asp) mutations were present exclusively in the SM resistant M. tuberculosis. Lys43Arg rpsL was the most predominant SMR mutations (69.6%) and prevailed among Beijing isolates (p<0.001). No SMR-related mutation in was found rrs. The combination of rpsL and gidB mutations provided 76.1% sensitivity for detecting SMR in M. tuberculosis Thai isolates. whiB7 was not responsible for SMR in SM resistant isolates lacking rpsL and rrs mutations. The significance of the three gidB mutations, 276A>C, 615A>G, and 330G>T, as lineage signatures for Beijing and EAI were underscored. This study identified 423G>A gidB as a novel sub-lineage marker for EAI6-BGD1. Conclusion: Our study suggested that the majority of SMR in M. tuberculosis Thai isolates were responsible by rpsL and gidB polymorphisms constantly providing the novel lineage specific makers.

• Tuberculosis and Respiratory Diseases
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• 제50권1호
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• pp.29-41
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• 2001

결핵환자에 있어 rpoB 유전자 염기서열 돌연변이로 인해 생겨나는 rifampin(RIF)내성은 화학요법치료에서 나타나는 다제내성의 표지자로서 많은 연구가 되어 있으며 rifabutin(RIB)은 이러한 RIF의 내성을 보이는 일부 점돌연변이에 대하여 감성 또는 내성을 보이는 것으로 보고되고 있다. 그러므로 본 연구에서는 mycobacteria rpoB 유전자의 특정 DNA 서열(17 bp)을 고정한 올리고뉴클레오티드 칩을 개발하여 rpoB 유전자의 점돌연변이으로 인한 RIF과 RIB의 감수성을 조사하고자 하였다. 방법 : 사용된 올리고뉴클레오티드 칩은 RIF 내성 프로브 및 RIB 감성 프로브를 포함하도록 고안되었으며, 각각의 돌연변이에 상응하는 야생형 프로브를 동일한 염기서열에서 선정하여 형광 시그날 세기의 직접비교에 의해 보다 정확한 탐지를 가능하게 하였다. 결과 : 15개의 임상 분리체를 검사한 결과 RIF 내성으로 밝혀진 돌연변이중 13개의 임상 분리체에서 RIB 감수성 돌연변이 종류를 판별할 수 있었다. 결론 : 올리고뉴레오티드 칩으로 rpoB 유전자에 대한 점돌연변이 연구는 결핵환자에 대한 RIF과 RIB 약제내성 유무를 판단케 함으로써 효과적인 화학요법치료를 가능케 할 것이며 기존 방법과 비교시 효율 및 재현성이 매우 높다고 판단되었다.

• Journal of Microbiology
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• 제35권3호
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• pp.172-176
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• 1997

The Mycobacterium tuberculosis clinical isolates in Korea, showing different drug resistances, were analyzed by comparing large restriction fragment (LRF) patterns produced y digestion of genomic DNA with infrequent-cutting endonucleases of SpeI, AsnI and pulsed-field gel electrophoresis (PFGE). SpeI and AsnI allowed with AsnI and SpeI, strains yielded an absolutely identical pattern for Korean type's mycobacteria even though they showed different drug resisstance. However, when three M. tuberculosis strains, showing drug resistance, were digested with XbaI, patterns were different from those of the other M. tuberculosis strians which are susceptible to drugs. This stuyd reveals that the comparison of chromosomal restriction patterns is very useful as an additional aid for the differentiation and identification of M. tuberculosis strains showing drug resistances.

• 대한의생명과학회지
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• 제11권4호
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• pp.465-471
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• 2005

Ofloxacin has antimycobacterial activity that possibly contributes a pivotal role in the second-line drug regimens that are used for the treatment of multidrug-resistant tuberculosis. However, in some communities, the resistance rate of Mycobacterium tuberculosis to this agent is surging. Therefore, a rapid and accurate method that can be used to determine the resistance of M tuberculosis to the ofloxacin can be very useful for effective treatment of the patients. As an effort to develop such a method, this study was set up to reveal general types of mutations that are related to ofloxacin resistance of M tuberculosis. From previous studies, it has been well known that ofloxacin resistance is associated with mutations in a gene encoding the gyrase A subunit protein. In this study, we obtained 43 ofloxacin-resistant and 50 ofloxacin-susceptible M tuberculosis clinical isolates from Masan National TB Hospital, and sequences of DNA fragment of 320 bp, region of gyrA corresponding to the ofloxacin resistance-determining region were analyzed. In brief, the results showed that a total of seven mutation types were found at gyrA. Theses mutations were all clustered within nucleotides 2574 to 2586 of the gyrA gene (codons 88 to 94). Codon 94 was the most frequently substituted site. Twenty-four of the 43 isolates had mutations at this position resulting in a total of five different types of amino acid changes $(Asp{\to}Ala,\;Asp{\to}Gly,\;Asp{\to}His,\;Asp{\to}Tyr,\;and\;Asp{\to}Asn)$. Five isolates contained a mutation at codon 90 resulting $Ala{\to}Val$ change. Four isolates had mutations at codon 91 causing a $Ser{\to}Pro$ change at this site. Two isolates contained a mutation at codon 88 and each of them resulted in different types of amino acid changes $(Gly{\to}Cys,\;Gly{\to}Ala)$. On the other hand, polymorphic site at codon 95 was found in both ofloxacin-resistant and ofloxacin-susceptible isolates. From these results, we concluded that the rate of mutations present in gyrA among ofloxacin-resistant M. tuberculosis in Korea is similar to the general rates of mutations found throughout the world. Subsequently, an oligonucleotide probe was designed based on the results of sequence analysis and was used to develop a dot blot hybridization assay system to determine ofloxacin-resistance of M tuberculosis. To evaluate this probe, dot-blot hybridization was carried out using other 57 clinical isolates, and the results showed that the dot-blot hybridization assay is good for detecting sequence alterations atgyrA gene.

• Tuberculosis and Respiratory Diseases
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• 제42권5호
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• pp.695-702
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• 1995

연구 배경: 결핵성 흉막삼출은 아직까지도 우리나라에서 가장 흔한 흉막삼출 원인으로 생각되고 있으나 기존의 검사로는 60% 정도의 확진만이 가능하다. 이에 보다 예민한 진단법의 개발이 요구되던 중 PCR의 발전으로 결핵성흉염의 진단에 도움을 줄 것으로 기대되었다. 여에 저자들은 PCR을 이용한 결핵성 흉막염 진단의 유용성을 알아보고자 하였다. 방법: 환자군으로 결핵성 흉막염으로 확진된 경우와 임상적으로 결핵성 흉막엽으로 의심이된 각각 7명의 선정하였고 대조군으로 결핵의 과거력이 없었던 7명의 악성종양 환자의 흉수를 각각 사용하여 PCR을 시행하였으며, PCR의 target은 IS6110 gene의 일부인 123bp DNA로 하였고 DNA추출은 Eisennach 방법(1991)을 변형하여 사용하였다. 결과: 1) PCR의 감수성검사에서 자외선 발광경하에서 50fg DNA에서 양성 임을 확인하였다. 2) 조직병리학적 및 미생물학적 방법으로 확진된 결핵성 흉막염 환자중 85.7%(6/7)에서 PCR양성을 나타내었고, 임상적으로 결핵성 흉막염이 의심된 환자중 71.5%(5/7)에서 PCR 양성을 나타내었다. 3) 대조군 7예는 모두 PCR 음성이었다. 결론: 이상의 결과로서 결핵성 흉막액의 진단에 있어서 IS6110을 이용한 PCR법은 고식적인 진단방법에 비하여 결핵균의 신속하고 정확한 진단을 가능하게 해주는 유용한 방법임을 알 수 있었으나 비용을 절감할 수 있고 오염을 줄일 수 있는 방법에 대한 연구가 더 필요하리라 생각된다.

• BMB Reports
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• 제34권4호
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• pp.342-346
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• 2001

The Mycobacterium bovis BCG panB gene, encoding ketopantoate hydroxymethyltransferase (KPHMT), was cloned from a ${\lambda}gt11$ genomic library and sequenced. The DNA sequence encodes a protein that contains 281 amino acid residues (M, 29,337) with a high similarity to the KPHMTs. Subcloning of a 846 by open reading frame (ORF), but not a 735 by ORF, into the vector pUC19 led to complementation of the panB mutant of Escherichia coli. The BCG pang gene was overexpressed in E. coli and the KPHMT purified to homogeneity The recombinant protein was further confirmed by an enzymatic assay.

• 대한미생물학회지
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• 제35권5_6호
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• pp.378-378
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• 2000

• Tuberculosis and Respiratory Diseases
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• 제53권6호
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• pp.635-643
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• 2002

연구배경 : 완전 자동화된 액체배양법과 기존의 고체배양법을 이용하여 폐결핵환자의 객담내 mycobacterium의 신속검출율 및 양성판정시까지의 시간을 비교하고자 하였다. 방 법 : 2002년 1월 1일부터 2002년 6월 30일 사이에 본원에 입원 혹은 외래에서 폐결핵으로 치료중인 환자 127명의 객담검체를 이용하였으며, 배양 중 오염된 28개의 검체(MB/BacT system 18개, L-J배지 14개, 4검체는 두 배양법 모두에서 오염)를 제외한 99명의 객담검체를 대상으로 하였다. 검체는 4% NaOH로 전처치한 후 4300rpm으로 20분간 원심분리한 후 멸균된 phosphate buffer(pH 6.8)를 첨가하여 2ml로 만들어 MB/BacT bottle에 0.5ml, Lowenstein-Jensen배지에 0.25ml를 접종한 후 $35-37^{\circ}C$에 6-12주간 배양하면서 균검출율과 양성판정시까지의 시간을 비교하였다. 결 과 : MB/Bact system과 L-J배양볍 중 어느 한 방법에서 균성장이 발견된 검제는 677B (67.7%) 였다. 두배양법 모두에서 균성장이 발견된 겸체는 5252.5%) 개였으며, MB/BacT에서만 발견된 검체는 15개 (15.2%) 였으나 L-J배양법에서만 발견된 경우는 없었다. 객담검체의 ZN염색법으로 항산균 집균도말겸사상 양성인 검체는 58개, 음성인 검체는 41개였다. 58개의 도말양성 검체 가운데 MB/BacT system에 양성을 보인 검체는 %개 (96.6%), L-J배지양성은 467B (79.3%) 였으며, 균성장 평균 발견시간은 각각 11.0일과 23.5일이었다. 41개의 도말음성검체 가운데 MB/BacT system에 양성을 보인 검체는 11개 (26.8%), L-J배지양성은 6개 (14.6%)였으며, 균성장 평균 발견시간은 각각 13.7일과 27.6일 이었다. 전체적으로 MB/BacT system과 L-J배양법에서 균성장 발견시 간은 각각 $13.3{\pm}8.3$일, $27.2{\pm}0.9$일이었다. 결 론 : 완전 자동화된 MB/BacT system은 L-J배지에 비해 mycobacterium의 검출율이 높고 발견시간이 단축되었다. MB/BacT는 방사성 물질을 사용하지 않는 장점이 있으므로 모든 검사실에 사용하기에 적당할 것으로 보이며, 균동정을 위하여 특이한 DNA probe와 병용하면 CDC가 권고한 결과보고 시간에 근접할 수 있을 것으로 생각되었다.

• Journal of Preventive Medicine and Public Health
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• 제45권1호
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• pp.1-7
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• 2012

Using three Austrian case studies, the variegated applications of molecular typing in today's public health laboratories are discussed to help illustrate preventive management strategies relying on DNA subtyping. DNA macrorestriction analysis by pulsed field gel electrophoresis has become the gold standard for subtyping of food borne pathogens like listeria, salmonella, campylobacter and Bacillus cereus. Using a Salmonella Mbandaka outbreak from the year 2010 as example, it is shown how the comparison of patterns from human isolates, food isolates, animal isolates and feed isolates can allow to identify and confirm a source of disease. An epidemiological connection between the simultaneous occurrence of tuberculosis in cattle and deer with cases of human tuberculosis due to Mycobacterium caprae in 2010 was excluded using mycobacterial interspersed repetitive units variable-number tandem repeats subtyping. Also in 2010, multilocus sequence typing with nonselective housekeeping genes, the so-called sequence based typing protocol, was used to elucidate connections between an environmental source (a hospital drinking water system) and a case of legionellosis. During the last decades, molecular typing has evolved to become a routine tool in the daily work of public health laboratories. The challenge is now no longer to simply type microorganisms, but to type them in a way that allows for data exchange between public health laboratories all over the world.

• Genomics & Informatics
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• 제12권4호
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• pp.276-282
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• 2014

The disease tuberculosis, caused by Mycobacterium tuberculosis (MTB), remains a major cause of morbidity and mortality in developing countries. The evolution of drug-resistant tuberculosis causes a foremost threat to global health. Most drug-resistant MTB clinical strains are showing resistance to isoniazid and rifampicin (RIF), the frontline anti-tuberculosis drugs. Mutation in rpoB, the beta subunit of DNA-directed RNA polymerase of MTB, is reported to be a major cause of RIF resistance. Amongst mutations in the well-defined 81-base-pair central region of the rpoB gene, mutation at codon 450 (S450L) and 445 (H445Y) is mainly associated with RIF resistance. In this study, we modeled two resistant mutants of rpoB (S450L and H445Y) using Modeller9v10 and performed a docking analysis with RIF using AutoDock4.2 and compared the docking results of these mutants with the wild-type rpoB. The docking results revealed that RIF more effectively inhibited the wild-type rpoB with low binding energy than rpoB mutants. The rpoB mutants interacted with RIF with positive binding energy, revealing the incapableness of RIF inhibition and thus showing resistance. Subsequently, this was verified by molecular dynamics simulations. This in silico evidence may help us understand RIF resistance in rpoB mutant strains.