• Journal of Microbiology and Biotechnology
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• v.17 no.5
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• pp.753-760
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• 2007

The diversity of Paenibacillus species was assessed in the rhizospheres of four cultivars of sorghum sown in Cerrado soil amended with two levels of nitrogen fertilizer(12 and 120 kg/ha). Two cultivars(IS 5322-C and IS 6320) demanded the higher amount of nitrogen to grow, whereas the other two(FBS 8701-9 and IPA 1011) did not. Using the DNA extracted from the rhizospheres, a Paenibacillus-specific PCR system based on the RNA polymerase gene(rpoB) was chosen for the molecular analyses. The resulting PCR products were separated into community fingerprints by DGGE and the results showed a clear distinction between cultivars. In addition, clone libraries were generated from the rpoB fragments of two cultivars(IPA 1011 and IS 5322-C) using both fertilization conditions, and 318 selected clones were sequenced. Analyzed sequences were grouped into 14 Paenibacillus species. A greater diversity of Paenibacillus species was observed in cultivar IPA 1011 compared with cultivar IS 5322-C. Moreover, statistical analyses of the sequences showed that the bacterial diversity was more influenced by cultivar type than nitrogen fertilization, corroborating the DGGE results. Thus, the sorghum cultivar type was the overriding determinative factor that influenced the community structures of the Paenibacillus communities in the habitats investigated.

• 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 Veterinary Science
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• v.19 no.6
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• pp.771-781
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• 2018

Staphylococcus aureus is one of the major pathogens causing bovine mastitis and foodborne diseases associated with dairy products. To determine the genetic relationships between human and bovine or bovine isolates of S. aureus, various molecular methods have been used. Previously we developed an rpoB sequence typing (RSTing) method for molecular differentiation of S. aureus isolates and identification of RpoB-related antibiotic resistance. In this study, we performed spa typing and RSTing with 84 isolates from mastitic cows (22 farms, 72 cows, and 84 udders) and developed a molecular prophage typing (mPPTing) method for molecular epidemiological analysis of bovine mastitis. To compare the results, human isolates from patients (n = 14) and GenBank (n = 166) were used for real and in silico RSTing and mPPTing, respectively. Based on the results, RST10-2 and RST4-1 were the most common rpoB sequence types (RSTs) in cows and humans, respectively, and most isolates from cows and humans clearly differed. Antibiotic resistance-related RSTs were not detected in the cow isolates. A single dominant prophage type and gradual evolution through prophage acquisition were apparent in most of the tested farms. Thus, RSTing and mPPTing are informative, simple, and economic methods for molecular epidemiological analysis of S. aureus infections.

• Tuberculosis and Respiratory Diseases
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• v.44 no.2
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• pp.251-263
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• 1997

Background : The emergence of multidrug-resistant strains of Mycobacterium tuberculosis presents a significant challange to the treatment and control of tuberculosis, and there is an urgent need to understand the mechanisms by which strains acquire multidrug resistance. Recent advances in molecular methods for the detection of M. tuberculosis genetic targets have approached the sensitivity of culture. Furthermore the prospect of determining resistance in mycobacteria at the nucleic acid level particulary to first-line drugs like rifampin, isoniazid has provided a glimps of the next generation of sensitivity test for M. tuberculosis. Previous studies in RMP resistant M. tuberculosis have shown that mutation in $\beta$subunit of RNA polymerase is main mechanism of resistance. Method : In this study, rpoB gene for the $\beta$subunit of RNA polymerase from M. tuberculosis of 42 cultured samples (32 were RMP resistant and 10 were sensitive cases) were isolated and characterised the mutations. Direct sequencing data were compared with the results of INNO-LiPA Line Probe Assay (LiPA, Innogenetics, Belgium), commercial RMP resistance detecting kit using reverse hybridization method. Results : All of the RMP resistant samples were revealed the presence of mutation by LiPA. In 22 samples (68.8%) out of 32 RMP resistant cases, the mutation types were confirmed by the positive signal at one of 4 mutation bands in the strip. The most frequent type was R5 (S531L) which were 17 cases (77.3%). Results of direct sequencing were identified the exact characteristics of 8 mutations which were not confirmed by LiPA. S522W type point mutation and 9 base pair deletion at codon 513~515 were new identified mutations for the first time. Conclusion : Mutations in rpoB gene is the main mechanism of RMP resistance in M. tuberculosis and LiPA is a very useful diagnostic tool for the early diagnosis of RMP resistance in M. tuberculosis.

• International Journal of Oral Biology
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• v.40 no.4
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• pp.205-210
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• 2015

Prevotella intermedia-specific quantitative real-time PCR (qPCR) primers were previously designed based on the nucleotide sequences of RNA polymerase ${\beta}$-subunit gene (rpoB). However, the several clinical strains isolated from Korean populations are not detectable by the qPCR primers. The purpose of this study was to develop new P. intermedia-specific qPCR primers based on the rpoB. The specificity of the primers was determined by conventional PCR with 12 strains of P. intermedia and 52 strains (52 species) of non-P. intermedia bacteria. The sensitivity of primers was determined by qPCR with serial dilutions of the purified genomic DNAs (40 ng to 4 fg) of P. intermedia ATCC $25611^T$. The data indicated that only P. intermedia strains were detected by the P intermedia-specific qPCR primers (RTPiF2/RTPiR2); in addition, as little as 40 fg of P. intermedia genomic DNA could be detected. These results suggest that these qPCR primers are useful in detecting P. intermedia from the bacterial infectious lesions including dental plaque and oral tissue lesions.

• Journal of fish pathology
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• v.22 no.3
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• pp.391-400
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• 2009

Genetic identification of 17 fish-derived Aeromonas strains was attempted using 5 housekeeping genes. 16S rRNA, gyrB, rpoD, dnaJ and recA genes from the 17 strains were amplified, and total of 85 amplicons were sequenced. DNA sequences of the strains and type strains of the 17 Aeromonas homology groups were used for genetic identification and phylogenetic analyses. None of the strains was identified as a single species using the 16S rRNA gene, showing the same identities (average = 99.7%) with several Aeromonas species. According to gyrB, rpoD, dnaJ, and recA, 9 strains and RFAS-1 used in this study were identified as A. hydrophila and A. salmonicida, respectively. However, the other strains were closely related to 2 or more Aeromonas species (i.e., A. salmonicida, A. veronii, A. jandaei, A. media and A. troda) depending on the genetic marker used. In this study, gyrB, rpoD, dnaJ and recA gene sequences proved to be advantageous over 16S rRNA for the identification of field Aeromonas isolates obtained from fish. However, there are discrepancies between analyses of different phylogenetic markers, indicating there are still difficulties in genetic identification of the genus Aeromonas using the housekeeping genes used in this study. Advantages and disadvantages of each housekeeping gene should be taken into account when the gene is used for identification of Aeromonas species.

• Journal of Microbiology and Biotechnology
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• v.8 no.3
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• pp.280-283
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• 1998

We have cloned the RNA polymerase sigma-like factors from a wide range of actinomycetes by using specific primers with the polymerase chain reaction (PCR). The specific oligonucleotide primers were designed on the basis of amino acid sequences of conserved regions from HrdA, B, D of Streptomyces griseus as well as from the rpoD box of many eubacteria. The consensus sequences were from the rpoD box and helix-turn-helix motif involved in -35 recognition. The designed primers were successfully applied to amplify the DNA fragments of the hrd homolog genes from 8 different strains of actinomycetes which produce a wide variety of important antibiotics. The 480 bp of the DNA fragment was amplified from all 8 strains, and it was identified as a part of hrdA and hrdB as we designed. The deduced amino acid sequence of PCR-amplified DNA fragments were highly homologous to those of other known RNA polymerase sigma factors of S. griseus and Streptomyces aureofaciens. Therefore, this study with specifically designed primers will support rapid cloning of the RNA polymerase sigma factors which recognize different classes of promoters from actinomycetes, and it will also be helpful in understanding the relationship of promoters and sigma factors leading to heterogeneity of RNA polymerases in actinomycetes.

• 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.

• Korean Journal of Microbiology
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• v.32 no.4
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• pp.264-270
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• 1994

A gene coding for a novel putative $\sigma$ factor of RNA polymerase has been identified from Streptomyces coelicolor A3(2) using Escherichia coli rpoS gene fragment as a probe. The 486 bp rpoS gene fragment was amplified from E. coli genomic DNA by PCR with two synthetic oligonucleotides, the sequences of which were deduced from the amino acid sequences in the regions 2.3 and 4.2 conserved among various bacterial factors. When E. coli genomic DNA fragments were hybridized with cloned rpoS probe, only one band corresponding to rpoS gene (3.2 kb PvuII fragment or 2.3 kb KpnI fragment) was detected. In S. coelicolor, however, two bands were detected both in PvuII digested DNA and SalI digested DNA. 3.5 kb PvuII fragment which binds the rpoS gene probe was cloned (pMS1) from the sublibrary, and the nucleotide sequences of 1.0 kb BamH'/HincII subclone (pBH2) was partially determined. The nucleotide sequences revealed extensive similarity to other $\sigma$ factor genes of S. coelicolor (hrdA, hrdB, hrdC, hrdD), S. aureofaciens (hrdA, hrdB, hrdC, hrdD), Synechococcus species, Pseudomonas aeruginosa, Stigmatella aurantiaca, and Anabaena species. The nucleotide sequences in regions 1.2 and 4 were compared with the corresponding regions of 5 known ${\sigma}$ factor genes of S. coelicolor by multiple alignment. It turned out that the cloned gene is most closely related to hrdA showing 88% amino acid similarity in region 1.2 and 75% in region 4.

• Tuberculosis and Respiratory Diseases
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• v.81 no.3
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• pp.222-227
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• 2018

Background: Rifampicin (RFP) is one of the principal first-line drugs used in combination chemotherapies against Mycobacterium tuberculosis, and its use has greatly shortened the duration of chemotherapy for the successful treatment of drug-susceptible tuberculosis. Compensatory mutations have been identified in rpoC that restore the fitness of RFP-resistant M. tuberculosis strains with mutations in rpoB. To investigate rpoC mutation patterns, we analyzed 93 clinical M. tuberculosis isolates from patients in South Korea. Methods: Drug-resistant mycobacterial isolates were cultured to determine their susceptibility to anti-tubercular agents. Mutations in rpoC were identified by sequencing and compared with the relevant wild-type DNA sequence. Results: In total, 93 M. tuberculosis clinical isolates were successfully cultured and tested for drug susceptibilities. They included 75 drug-resistant tuberculosis species, of which 66 were RFP-resistant strains. rpoC mutations were found in 24 of the 66 RFP-resistant isolates (36.4%). Fifteen different types of mutations, including single mutations (22/24, 91.7%) and multiple mutations (2/24, 8.3%), were identified, and 12 of these mutations are reported for the first time in this study. The most frequent mutation involved a substitution at codon 452 (nt 1356) resulting in amino acid change F452L. Conclusion: Fifteen different types of mutations were identified and were predominantly single-nucleotide substitutions (91.7%). Mutations were found only in dual isoniazid- and RFP-resistant isolates of M. tuberculosis. No mutations were identified in any of the drug-susceptible strains.