Patterns of rpoC Mutations in Drug-Resistant Mycobacterium tuberculosis Isolated from Patients in South Korea.

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.

Meta-analysis of genome-wide SNP- and pathway-based associations for facets of neuroticism.

Neuroticism is a heritable personality trait that is comprised of distinct sub-factors, or facets. Sub-factors of neuroticism are linked to different emotional states or psychiatric symptoms and studying the genetic variants associated with these facets may help reveal the biological mechanisms underlying psychiatric disorders. In the present study, a meta-analysis of genome-wide association studies for six facets of neuroticism was performed in 5584 participants from three cohorts. Additionally, a Gene Set Enrichment Analysis was conducted to find biological pathways associated with each facet. Six neuroticism facets (N1: anxiety, N2: angry hostility, N3: depression, N4: self-consciousness, N5: impulsivity and N6: vulnerability) were assessed using the Korean version of the Revised NEO Personality Inventory. In the single-nucleotide polymorphism-based analysis, results showed genome-wide significance for N2 within the MIR548H3 gene (rs1360001, P=4.14 × 10-9). Notable genes with suggestive associations (P<1.0 × 10-6) were ITPR1 for N1, WNT7A for N2, FGF10 and FHIT for N3, DDR1 for N4, VGLL4 for N5 and PTPRD for N6. In the pathway-based analysis, the axon guidance pathway was identified to be associated with multiple facets of neuroticism (N2, N4 and N6). The focal adhesion and extracellular matrix receptor interaction pathways were significantly associated with N2 and N3. Our findings revealed genetic influences and biological pathways that are associated with facets of neuroticism.

pncA Mutations in the Specimens from Extrapulmonary Tuberculosis.

BACKGROUND: Pyrazinamide (PZA) is an effective antitubercular drug that becomes toxic to Mycobacterium tuberculosis when converted to pyrazinoic acid by pyrazinamidase (PZase), encoded by mycobacterial pncA. A strong association was noted between the loss of PZase activity and PZA resistance. The causative organisms in extrapulmonary tuberculosis are rarely cultured and isolated. To detect pncA mutations in specimens from extrapulmonary tuberculosis as confirmative diagnosis of mycobacterial infection and alternative susceptibility test to PZA. METHODS: Specimens were collected from clinically proven extrapulmonary tuberculosis. pncA was sequenced and compared with wild-type pncA. RESULTS: pncA from 30 specimens from 23 donors were successfully amplified (56.6% in specimens, 59% in donors). Six mutations in pncA were detected (20.0% in amplified specimens, 26.1% in specimen donors) at nucleotide positions of 169, 248 and 419. The mutation at position 169 results in substitution of aspartic acid for histidine, a possible allelic variation of M. bovis that have intrinsic PZA resistance. The mutation at position 248 changes proline into arginine and that at position 419, arginine into histidine. CONCLUSION: DNA-based diagnosis using pncA may be simultaneously useful for the early diagnosis of mycobacterial infection and the rapid susceptibility to PZA in extrapulmonary tuberculosis. A potential implication of pncA allelic variation at 169 might be suggested as a rapid diagnostic test for M. bovis infection or Bacille Calmette-Guérin (BCG) reactivation.

Analysis of population structure among Korean and Japanese Legionella pneumophila isolates using hsp60 sequences.

The population structure of Korean (150 strains) and Japanese (92 strains) Legionella pneumophila isolates along with 18 reference strains were investigated using hsp60 sequence (1647 bp) analysis. Twelve clonal subgroups (hsP-I to hsP-X and hsF-I and hsF-II) were designated on the hsp60 tree, inferred from representative sequences using the neighbor-joining method. Some of the isolates showed unique subgroups depending on the source of isolates, including hsP-I, hsF-I, and hsF-II from cooling tower water, and subgroups hsP-VIII and hsP-X from circulating hot water bath. These subgroups may be useful for epidemiological studies to chase or specify sources of infection in Korea and Japan.

Mycobacterium massiliense is differentiated from Mycobacterium abscessus and Mycobacterium bolletii by erythromycin ribosome methyltransferase gene (erm) and clarithromycin susceptibility patterns.

Erythromycin ribosome methyltransferase gene (erm) sequences of Mycobacterium massiliense and Mycobacterium bolletii isolates were newly investigated. Forty nine strains of M. massiliense that were analyzed in the present study had a deleted erm(41). Due to a frame-shift mutation, large deletion, and truncated C-terminal region, the Erm(41) of M. massiliense had only 81 amino acids encoded by 246 nucleotides. Corresponding to these findings, most of the M. massiliense isolates (89.8%) were markedly clarithromycin susceptible, but resistant strains invariably had a point mutation at the adenine (A(2058) or A(2059)) in the peptidyltransferase region of the 23S rRNA gene, which is quite different from Mycobacterium abscessus and M. bolletii. In addition, erm(41) sequences of M. massiliense were more conserved than those of M. abscessus and M. bolletii. The results of species identification using erm(41) showed concordant results with those of multi-locus sequence analysis (rpoB, hsp65, sodA and 16S-23S ITS) where there were originally inconsistent results between rpoB and hsp65 sequence analysis in previous research. Therefore, erm(41) PCR that was used in the present study can be efficiently used to simply differentiate M. massiliense from M. abscessus and M. bolletii.

Differentiation of Bifidobacterium species using partial RNA polymerase {beta}-subunit (rpoB) gene sequences.

Partial RNA polymerase ß-subunit gene (rpoB) sequences (315 bp) were determined and used to differentiate the type strains of 23 species of the genus Bifidobacterium. The sequences were compared with those of the partial hsp60 (604 bp) and 16S rRNA genes (1475 or 1495 bp). The rpoB gene sequences showed nucleotide sequence similarities ranging from 84.1 % to 99.0 %, while the similarities of the hsp60 sequences ranged from 78.5 % to 99.7 % and the 16S rRNA gene sequence similarities ranged from 89.4 % to 99.2 %. The phylogenetic trees constructed from the sequences of these three genes showed similar clustering patterns, with the exception of several species. The Bifidobacterium catenulatum-Bifidobacterium pseudocatenulatum, Bifidobacterium pseudolongum subsp. pseudolongum-Bifidobacterium pseudolongum subsp. globosum and Bifidobacterium gallinarum-Bifidobacterium pullorum-Bifidobacterium saeculare groups were more clearly differentiated in the partial rpoB and hsp60 gene sequence trees than they were in the 16S rRNA gene tree. Based on sequence similarities and tree topologies, the newly determined rpoB gene sequences are suitable molecular markers for the differentiation of species of the genus Bifidobacterium and support various other molecular tools used to determine the relationships among species of this genus.

Proportions of Mycobacterium massiliense and Mycobacterium bolletii strains among Korean Mycobacterium chelonae-Mycobacterium abscessus group isolates.

Korean isolates of the Mycobacterium chelonae-Mycobacterium abscessus group, which had been isolated from two different hospitals in South Korea, were identified by PCR restriction analysis (PRA) and comparative sequence analysis of 16S rRNA genes, rpoB, and hsp65 to evaluate the proportion of four closely related species (M. chelonae, M. abscessus, M. massiliense, and M. bolletii). Of the 144 rapidly growing mycobacterial strains tested, 127 strains (88.2%) belonged to the M. chelonae-M. abscessus group. In this group, M. chelonae, M. abscessus, M. massiliense, and M. bolletii accounted for 0.8% (n = 1), 51.2% (n = 65), 46.5% (n = 59), and 1.6% (n = 2), respectively. Two isolates which showed discordant results, M. massiliense by rpoB sequence analysis and M. abscessus by hsp65 sequence analysis, were finally identified as M. massiliense based on the additional analysis of sodA and the 16S-23S internal transcribed spacer. M. abscessus group I isolates previously identified by hsp65 PRA were all found to be M. abscessus, whereas group II isolates were further identified as M. massiliense or M. bolletii by sequencing of rpoB and hsp65. Smooth, rough, or mixed colonies of both M. abscessus and M. massiliense isolates were observed. M. massiliense strains that were highly resistant to clarithromycin had a point mutation at the adenine at position 2058 (A(2058)) or 2059 (A(2059)) in the peptidyltransferase region of the 23S rRNA gene.

Outbreak of Mycobacterium massiliense infection associated with intramuscular injections.

Twelve strains of a rapidly growing Mycobacterium species were isolated from an outbreak associated with intramuscular injections of an antimicrobial agent and were identified by comparative sequence analysis of rpoB and hsp65. These isolates were identified as Mycobacterium massiliense (100% similarity).

Detection and identification of Mycobacterium tuberculosis in joint biopsy specimens by rpoB PCR cloning and sequencing.

Osteoarticular tuberculosis (OAT) is an extrapulmonary tuberculosis and accounts for 1 to 3% of all tuberculosis cases. We used an rpoB PCR-plasmid TA cloning-sequencing method to detect and identify tubercle bacilli in surgical specimens from patients suspected of having OAT. By comparing the similarities of the rpoB sequences determined with those in GenBank, Mycobacterium tuberculosis was detected in 23 of 43 samples. Three of the 23 positive samples had mutations at codon 531, which are commonly observed in rifampin-resistant M. tuberculosis strains. Our results suggest that the rpoB PCR-TA cloning-sequencing method developed, which detects M. tuberculosis and which simultaneously determines its rifampin susceptibility, can also be used efficiently for the diagnosis of OAT.

Identification of Mycobacterium tuberculosis by PCR-linked reverse hybridization using specific rpoB oligonucleotide probes.

A reverse probe hybridization method using two different Mycobacterium tuberculosis-specific rpoB DNA probes in combination was evaluated for the identification of M. tuberculosis culture isolates. Among the 384 isolates tested, 354 strains were identified as M. tuberculosis, which included 37 rifampin-resistant strains, and 30 were nontuberculous mycobacteria (NTM). This result was in accord with partial rpoB sequence analysis and IS6110 polymerase chain reaction (PCR) results, but not with the results of biochemical testing, which produced two false negative results. Because of its high level of sensitivity and specificity, we suggest that M. tuberculosis-specific rpoB probes immobilized on micro-titer well plates or on other solid matrixes can be used efficiently for the rapid and convenient identification of M. tuberculosis.

Alanine-threonine polymorphism of Helicobacter pylori RpoB is correlated with differential induction of interleukin-8 in MKN45 cells.

Geographical differences in the genetic diversity of Helicobacter pylori isolates were examined by analyzing rpoB sequences. An extremely high level of allelic diversity among H. pylori strains was found. The rpoB sequences of Asian and non-Asian (North and South American, European, and South African) strains were found to differ. An amino acid polymorphism (alanine and threonine RpoB types) was found at the 497th residue by deduced amino acid analysis. RpoB with a threonine residue (RpoB(Thr)) was uniquely present in East Asian countries, and two-thirds of the H. pylori isolate population in this region was RpoB(Thr); however, this type was rare or absent in Western countries, where RpoB(Ala) predominated. RpoB(Thr) strains induced a much larger amount of interleukin-8, a chemokine that plays an important role in chronic inflammation, than RpoB(Ala) strains in cultured MKN45 cells.

Simultaneous identification of rifampin-resistant Mycobacterium tuberculosis and nontuberculous mycobacteria by polymerase chain reaction-single strand conformation polymorphism and sequence analysis of the RNA polymerase gene (rpoB).

Interspecies variations and mutations associated with rifampin resistance in rpoB of Mycobacterium allow for the simultaneous identification of rifampin-resistant Mycobacterium tuberculosis and nontuberculous mycobacteria by PCR-SSCP analysis and PCR- sequencing. One hundred and ten strains of rifampin-susceptible M. tuberculosis, 14 strains of rifampin-resistant M. tuberculosis, and four strains of the M. avium complex were easily identified by PCR-SSCP. Of another seven strains, which showed unique SSCP patterns, three were identified as rifampin-resistant M. tuberculosis and four as M. terrae complex by subsequent sequence analysis of their rpoB DNAs (306 bp). These results were concordant with those obtained by susceptibility testing, biochemical identification, and 16S rDNA sequencing.

Differential identification of Mycobacterium tuberculosis complex and nontuberculous mycobacteria by duplex PCR assay using the RNA polymerase gene (rpoB).

A novel duplex PCR method that can amplify the 235- and 136-bp rpoB DNAs of Mycobacterium tuberculosis complex and nontuberculous mycobacteria (NTM), respectively, with two different sets of primers was used to differentially identify 44 reference strains and 379 clinical isolates of mycobacteria in a single-step assay. Showing 100% sensitivity and specificity, the duplex PCR method could clearly differentiate M. tuberculosis complex and NTM strains. In addition, restriction fragment length polymorphism analysis and direct sequencing of the amplicon of NTM could be used to supplement species identification.

Application of RNA polymerase beta-subunit gene (rpoB) sequences for the molecular differentiation of Legionella species.

The nucleotide sequences of the partial rpoB gene were determined from 38 Legionella species, including 15 serogroups of Legionella pneumophila. These sequences were then used to infer the phylogenetic relationships among the Legionella species in order to establish a molecular differentiation method appropriate for them. The sequences (300 bp) and the phylogenetic tree of rpoB were compared to those from analyses using 16S rRNA gene and mip sequences. The trees inferred from these three gene sequences revealed significant differences. This sequence incongruence between the rpoB tree and the other trees might have originated from the high frequency of synonymous base substitutions and/or from horizontal gene transfer among the Legionella species. The nucleotide variation of rpoB enabled more evident differentiation among the Legionella species than was achievable by the 16S rRNA gene and even by mip in some cases. Two subspecies of L. pneumophila (L. pneumophila subsp. pneumophila and subsp. fraseri) were clearly distinguished by rpoB but not by 16S rRNA gene and mip analysis. One hundred and five strains isolated from patient tissues and environments in Korea and Japan could be identified by comparison of rpoB sequence similarity and phylogenetic trees. These results suggest that the partial sequences of rpoB determined in this study might be applicable to the molecular differentiation of Legionella species.

Population genetic structure of Legionella pneumophila inferred from RNA polymerase gene (rpoB) and DotA gene (dotA) sequences.

The population structure of Legionella pneumophila was studied by using partial RNA polymerase gene (rpoB) and DotA gene (dotA) sequences. Trees inferred from rpoB sequences showed that two subspecies of L. pneumophila, Legionella pneumophila subsp. pneumophila and Legionella pneumophila subsp. fraseri, were clearly separated genetically. In both rpoB and dotA trees, 79 Korean isolates used in this study constituted six clonal populations, four of which (designated subgroups P-I to P-IV) were identified in L. pneumophila subsp. pneumophila and two of which (designated subgroups F-I and F-II) were identified in L. pneumophila subsp. fraseri. Although the relationships among subgroups were not identical, such subgrouping was congruent between the rpoB and dotA trees. Type strains of several serogroups did not belong to any subgroup, presumably because isolates similar to these strains were not present among our local sample of the population. There was evidence that horizontal gene transfer or recombination had occurred within L. pneumophila. Contrary to the phylogeny from rpoB and the taxonomic context, subgroups P-III and P-IV of L. pneumophila subsp. pneumophila proved to be closely related to those of L. pneumophila subsp. fraseri or showed a distinct clustering in the dotA tree. It can be inferred that dotA of subgroups P-III and P-IV has been transferred horizontally from other subspecies. The diverse distribution of serogroup 1 strains through the gene trees suggests that surface antigen-coding genes that determine serogroup can be exchanged. Thus, it can be inferred that genetic recombination has been important in the evolution of L. pneumophila.