[Show abstract][Hide abstract] ABSTRACT: Recently, we introduced a novel peptide nucleic acid (PNA) multi-probe real time PCR method targeting the hsp65 gene (hsp65 PNA RT-PCR) to distinguish Mycobacterium abscessus groups.
Here, we evaluated the usefulness of the hsp65 PNA RT-PCR for the direct identification of the M. abscessus group at the subspecies and genotype levels from sputa samples. The method was applied to total sputa DNA from 60 different patients who were identified as having mycobacterial infections via rpoB PCR restriction analysis of the same cultures.
The hsp65 PNA RT-PCR method had higher sensitivity than the multi-probe real-time PCR assay targeting hsp65 (HMPRT-PCR) for the detection of M. abscessus from sputum [96.7 % (29/30 samples) vs. 70 % (21/30 samples); 100 % specificity].
These results suggest that the PNA-based method is feasible for the detection of M. abscessus members not only from cultures but also directly from sputa.
[Show abstract][Hide abstract] ABSTRACT: From the whole blood of Korean native cattle, Hanwoo (Bos taurus coreanae), a previously undescribed, rapidly growing scotochromogenic Mycobacterium isolate is reported. Its 16S rRNA sequence, and other three different gene sequences (hsp65, recA and rpoB) were unique and the phylogenetic analysis based on 16S rRNA sequence (1420 bp) placed the organism into the rapidly growing Mycobacterium group close to Mycobacterium smegmatis (98.5 % of sequence similarity). But, phylogenetic analyses based on three different gene sequences (hsp65, recA and rpoB) revealed its distinct location from branch of rapidly growing species. Culture and biochemical characteristics were generally similar to those of Mycobacterium fortuitum. Unique MALDI-TOF MS profiles of lipids, unique fatty acid profile, unique mycolic acids profiles and a low DNA-DNA relatedness to M. fortuitum (23.6 %) and M. smegmatis (39.7 %) strongly supported the taxonomic status of this strain as a novel rapidly growing mycobacteria. The type strain is strain QIA-38T and named as Mycobacterium anyangense (=JCM 30275T=KCTC 29443T).
International Journal of Systematic and Evolutionary Microbiology 04/2015; 65(7). DOI:10.1099/ijs.0.000255 · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Mycobacterium-Escherichia coli shuttle vector system, equipped with the pAL5000 replicon, is widely used for heterologous gene expression and gene delivery in mycobacteria. Despite its extensive use, this system has certain limitations, which has led to the development of alternative mycobacterial vector systems. The present study describes the molecular structure and expression profiles of a novel 18-kb linear plasmid, pMyong2, from Mycobacterium yongonense. Sixteen open reading frames and a putative origin of replication were identified, and the compatibility of the pMyong2 and pAL5000 vector systems was demonstrated. In recombinant Mycobacterium smegmatis (rSmeg), the pMyong2 vector system showed a copy number that was approximately 37 times greater than that of pAL5000. Furthermore, pMyong2 increased the mRNA and protein expression of the human macrophage migration inhibitory factor (hMIF) over pAL5000 levels by approximately 10-fold and 50-fold, respectively, demonstrating the potential utility of the pMyong2 vector system in heterologous gene expression in mycobacteria. Successful delivery of the EGFP gene into mammalian cells via rSmeg carrying the pMyong2 vector system was also observed, demonstrating the feasibility of this system for DNA delivery. In conclusion, the pMyong2 vector system could be effectively used not only for the in vivo delivery of recombinant protein and DNA but also for mycobacterial genetic studies as an alternative or a complement to the pAL5000 vector system.
PLoS ONE 03/2015; 10(3):e0122897. DOI:10.1371/journal.pone.0122897 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Combinatorial molecular taxonomic approaches targeting 3 genes, 16S rRNA (1.2-1.3kbp), hsp65 (603-bp), and rpoB genes (711-bp) were applied to 43 non-tuberculous mycobacteria (NTM) strains isolated from a Korean native cattle from bronchial lymph nodes and lung, Hanwoo (Bos taurus coreanae) in South Korea. Of 43 NTM isolates, Mycobacterium avium complex strains (MAC) were isolated with the highest frequency (31 strains, 72.1%). Contrary to other reports, M. intracellulare strains (23 strains, 53.5%) of MACs were more prevalent than M. avium strains (8 strains, 18.6%). Further separation of isolated M. intracellulare into genotype level by hsp65 analysis showed that isolates of the HG-1 genotype (60.9%, 14/23 isolates), known to be specific to Korean patients, was more prevalent than the HG-2 type (17.4%, 4/23 strains), which include the type strain, M. intracellulare ATCC 13950(T). Compared to NTM infections of Korean human patients, the pronounced difference found in this study is that no M. abscessus infections in Hanwoo were found. In conclusion, our data showed that the isolated species frequency of NTMs, particularly MACs from Hanwoo, was very comparable to that obtained from Korean human infection, suggesting that humans and Korean native cattle may share common environmental sources for NTM infections.
[Show abstract][Hide abstract] ABSTRACT: Recently, we introduced the complete genome sequence of Mycobacterium massiliense clinical isolates, Asan 50594 belonging to Type II genotype with rough colony morphology. Here, to address the issue of whether the rough colony morphotype of M. massiliense Type II genotype is genetically determined or not, we compared polymorphisms of the glycopeptidolipid (GPL) gene locus between M. massiliense Type II Asan 50594 and other rapidly growing mycobacteria (RGM) strains via analysis of genome databases.
We found deletions of 10 genes (24.8 kb), in the GPL biosynthesis related gene cluster of Asan 50594 genome, but no deletions in those of other smooth RGMs. To check the presence of deletions of GPL biosynthesis related genes in Mycobacterium abscessus - complex strains, PCRs targeting 12 different GPL genes (10 genes deleted in Asan 50594 genome as well as 2 conserved genes) were applied into 76 clinical strains of the M. abscessus complex strains [54 strains (Type I: 33, and Type II: 21) of M. massiliense and 22 strains (rough morphoype: 11 and smooth morphotype: 11) of M. abscessus]. No strains of the Type II genotype produced PCR amplicons in a total of 10 deleted GPL genes, suggesting loss of GPL biosynthesis genes in the genome of M. massiliense type II genotype strains.
Our data suggested that the rough colony morphotype of the M. massiliense Type II genotype may be acquired via deletion events at the GPL gene locus for evolutionary adaptation between the host and pathogen.
[Show abstract][Hide abstract] ABSTRACT: A previously undescribed, a slow growing scoto-chromogenic mycobacterial species was isolated from a patient with pulmonary infections during the hsp65 sequence based identification of Korean clinical isolates. Growth characteristics, acid fastness, and results of 16S rDNA sequencing supported the placement of this species into the genus Mycobacterium. Phenotypically, this strain was generally similar to Mycobacterium gordonae, however, of particular interest, the optimal growth temperature of strain 49061T was 25 to 30 , and it is not able to grow at 37 on 7H10 agar slant. Its 16S rRNA sequence was unique and the phylogenetic analysis based on 16S rRNA sequence (1393 bp) placed the organism into the slow growing Mycobacterium group close to Mycobacterium gordonae (99.0 % of sequence similarity). Unique MALDI-TOF MS profiles of lipids, phylogenetic analysis based on other two different gene sequences (hsp65 and rpoB) and a low DNA-DNA relatedness (46.52 ± 0.7) strongly supported the taxonomic status of this strain as a distinct species from M. gordonae. The type strain is strain 49061T (=JCM 18565T=KCTC 29126T).
International Journal of Systematic and Evolutionary Microbiology 09/2013; 64(Pt 1). DOI:10.1099/ijs.0.051540-0 · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report the complete genome sequence of the Mycobacterium massiliense clinical strain Asan 50594, which was grouped into the M. massiliense type II genotype, isolated from a Korean patient. This genome sequence will serve as a valuable reference for understanding the disparity in virulence and epidemiological traits between strains belonging to the Mycobacterium abscessus complex.
[Show abstract][Hide abstract] ABSTRACT: Recently, a novel species, Mycobacterium yongonense (DSM 45126(T)), was introduced and while it is phylogenetically related to Mycobacterium intracellulare, it has a distinct RNA polymerase β-subunit gene (rpoB) sequence that is identical to that of Mycobacterium parascrofulaceum, which is a distantly related scotochromogen, which suggests the acquisition of the rpoB gene via a potential lateral gene transfer (LGT) event. The aims of this study are to prove the presence of the LGT event in the rpoB gene of the M. yongonense strains via multilocus sequence analysis (MLSA). In order to determine the potential of an LGT event in the rpoB gene of the M. yongonense, the MLSA based on full rpoB sequences (3447 or 3450 bp) and on partial sequences of five other targets [16S rRNA (1383 or 1395 bp), hsp65 (603 bp), dnaJ (192 bp), recA (1053 bp), and sodA (501 bp)] were conducted. Incongruences between the phylogenetic analysis of the full rpoB and the five other genes in a total of three M. yongonense strains [two clinical strains (MOTT-12 and MOTT-27) and one type strain (DSM 45126(T))] were observed, suggesting that rpoB gene of three M. yongonense strains may have been acquired very recently via an LGT event from M. parascrofulaceum, which is a distantly related scotochromogen.
PLoS ONE 01/2013; 8(1):e51846. DOI:10.1371/journal.pone.0051846 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A previously undescribed, slowly growing, non-chromogenic Mycobacterium strain (strain 299T) was isolated from the sputum sample of a patient with a symptomatic pulmonary infection. Phenotypically, strain 299T was generally similar to Mycobacterium koreense DSM 45576T and M. triviale ATCC 23292T. The 16S rRNA gene sequence of strain 299T was similar to those of M. koreense DSM 45576T (AY734996, 99.5 % similarity); however, it differed substantially from that of M. triviale ATCC 23292T (X88924, 98.2 %). Phylogenetic analysis based on 16S rRNA gene sequence showed that the strain 299T was clustered together with M. koreense DSM 45576T and M. triviale ATCC 23292T, supported by the high bootstrapping values (99 %). Unique mycolic acid profiles and phylogenetic analysis based on two different chronometer molecules, and the hsp65 and rpoB genes, strongly supported the taxonomic status of this strain as representing a distinct species. These data support the conclusion that strain 299T represents a novel mycobacterial species, for which the name M. parakoreense sp. nov. is proposed. The type strain is the strain 299T (=DSM 45575T=KCTC 19818T).
International Journal of Systematic and Evolutionary Microbiology 11/2012; 63(Pt 6). DOI:10.1099/ijs.0.045070-0 · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous studies have proved the presence of several distinct types of mutations in hepatitis B virus (HBV) infections, which are related to the progression of liver disease. However, few reports have detailed the mutation frequencies and mutation patterns in the precore/core (preC/C) region, which are based on the clinical status and HBeAg serostatus. Our aim in this study is to investigate the relationships between the preC/C mutations and clinical severity or HBeAg serostatus from patients chronically infected with HBV genotype C. A total of 70 Korean chronic patients, including 35 with hepatocellular carcinoma (HCC), participated in this study. HBV genotyping and precore/core mutations were analyzed by direct sequencing. All patients were confirmed to have genotype C infections. Mutations in the C region were distributed in a non-random manner. In particular, mutations in the MHC class II restricted region were found to be significantly related to HCC. Six (preC-W28*, C-P5H/L/T, C-E83D, C-I97F/L, C-L100I and C-Q182K/*) and seven types (preC-W28*, preC-G29D, C-D32N/H, C-E43K, C-P50A/H/Y, C-A131G/N/P and C-S181H/P) of mutations in the preC/C region were found to be related to HCC and to affect the HBeAg serostatus, respectively. In conclusion, our data indicated that HBV variants in the C region, particularly in the MHC class II restricted region, may contribute to the progress of HCC in chronic patients infected with genotype C. In addition, we found several distinct preC/C mutations in the Korean chronic cohort, which affect the clinical status of HCC and HBeAg serostatus of patients infected with genotype C.
PLoS ONE 10/2012; 7(10):e47372. DOI:10.1371/journal.pone.0047372 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: So far, genetic diversity among strains within Mycobacterium massiliense has rarely been studied. To investigate the genetic diversity among M. massiliense, we conducted phylogenetic analysis based on hsp65 (603-bp) and rpoB (711-bp) sequences from 65 M. massiliense Korean isolates. We found that hsp65 sequence analysis could clearly differentiate them into two distinct genotypes, Type I and Type II, which were isolated from 35 (53.8%) and 30 patients (46.2%), respectively. The rpoB sequence analysis revealed a total of four genotypes (R-I to R-IV) within M. massiliense strains, three of which (R-I, R-II and R-III) correlated with hsp65 Type I, and other (R-IV), which correlated with Type II. Interestingly, genotyping by the hsp65 method agreed well with colony morphology. Despite some exceptions, Type I and II correlated with smooth and rough colonies, respectively. Also, both types were completely different from one another in terms of MALDI-TOF mass spectrometry profiles of whole lipid. In addition, we developed PCR-restriction analysis (PRA) based on the Hinf I digestion of 644-bp hsp65 PCR amplicons, which enables the two genotypes within M. massiliense to be easily and reliably separated. In conclusion, two distinct hsp65 genotypes exist within M. massiliense strains, which differ from one another in terms of both morphology and lipid profile. Furthermore, our data indicates that Type II is a novel M. massiliense genotype being herein presented for the first time. The disparity in clinical traits between these two hsp65 genotypes needs to be exploited in the future study.
PLoS ONE 09/2012; 7(6):e38420. DOI:10.1371/journal.pone.0038420 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Here we report the complete genome sequence of the Mycobacterium intracellulare clinical strain MOTT-36Y, previously grouped into the INT5 genotype among the 5 genotypes of M. intracellulare. This genome sequence will serve as a valuable reference for understanding the disparity in virulence and epidemiologic traits between M. intracellulare-related strains.
Journal of bacteriology 08/2012; 194(15):4141-2. DOI:10.1128/JB.00752-12 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Microbiology and Immunology 06/2012; 56(8):572-8. DOI:10.1111/j.1348-0421.2012.00474.x · 1.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Here, we report the complete genome sequence of the Mycobacterium intracellulare clinical strain MOTT-64, previously grouped into the INT1 genotype among five genotypes of M. intracellulare. This genome sequence will serve as a valuable reference for understanding the disparity in the virulence and epidemiologic traits among M. intracellulare genotypes.
Journal of bacteriology 06/2012; 194(12):3268. DOI:10.1128/JB.00471-12 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Specimens were collected from clinically proven extrapulmonary tuberculosis. pncA was sequenced and compared with wild-type pncA.
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.
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.