Aims: To evaluate sensitivity and specificity of the newly developed PaxView TB/NTM MPCR-ULFA Kit. Study Design: Compared with the licensed AdvanSure TB/NTM real-time PCR. Place and Duration of Study: SCL and PaxGenBio in Gyeonggi-do, Korea, between August 2018 and May 2019. Methodology: In this study, 350 specimens including sputum, bronchial washing, body fluid, tissue, urine, and cerebrospinal fluid were examined to evaluate the performance of the PaxView TB/NTM MPCR-ULFA Kit compared to results of the currently licensed AdvanSure TB/NTM real-time PCR (LG Chem, Korea). Results: Compared to the AdvanSure TB/NTM real-time PCR, the PaxView TB/NTM MPCR-ULFA Kit test was found to possess a 100% sensitivity. In other words, all 140 MTB and 61 non-tuberculous mycobacteria (NTM) specimens that tested positive with the AdvanSure TB/NTM real-time PCR also tested positive with the PaxView TB/NTM MPCR-ULFA Kit. However, the specificity of the later kit found to be 97.9% (146/149; 95% CI 95.6–100.0), meaning that out of 149 MTB/NTM specimens that tested negative with the AdvanSure TB/NTM real-time PCR, 146 were identified as MTB/NTM-negative according to the PaxView TB/NTM MPCR-ULFA Kit. Nonetheless, the overall agreement between the two diagnostic tools was 99.1% (347/350; 95% CI 98.1– 100.0) and the kappa value was 0.982 (350; 95% CI 0.968 – 0.995), meaning that the two diagnostic tools rendered almost identical results. Conclusion: The PaxView TB/NTM MPCR-ULFA Kit could be useful to identify MTB and NTM in resource-limited countries, as this procedure is far more cost-effective than real-time PCR and convenient than conventional gel electrophoresis approaches.

BACKGROUND:Mycobacterium abscessus group belongs to a group of rapidly growing mycobacteria (RGM) and, following Mycobacterium avium complex, is the second most common pathogen responsible for lung disease caused by nontuberculous mycobacteria (NTM). Clarithromycin is known to be the key drug in the treatment of M. abscessus group disease, but a high failure rate of treatment response is reported due to clarithromycin inducible resistance.METHODS:Using the results from a clarithromycin susceptibility test we examined the proportion of clarithromycin inducible resistant M. abscessus (sensu stricto; hereafter referred to as M. abscessus) clinical strains. Also, we attempted to detect the clarithromycin resistant strains, using the amplification refractory mutation system-PCR (ARMS-PCR) and real-time PCR methods for rapid detection of single-nucleotide polymorphisms (SNPs) at position 28 (T or C) of the erm(41) gene of M. abscessus leading to resistance to clarithromycin.RESULTS:Of the 157 M. abscessus clinical strains, clarithromycin susceptible, resistant, and inducible resistant strains accounted for 10.83% (n = 17), 22.29% (n = 35), and 66.88% (n = 105), respectively. Clarithromycin resistant strains were able to separate from clarithromycin susceptible strains by ARMS-PCR and real-time PCR identical to DNA sequence analysis.CONCLUSION:Most M. abscessus clinical strains in Korea are resistant to clarithromycin, and ARMS-PCR and real-time PCR are useful tools for the rapid detection of single-nucleotide polymorphisms (SNPs) at position 28 of the erm(41) gene.

Mycobacterium massiliense (Mmass) is an emerging, rapidly growing mycobacterium (RGM) that belongs to the M. abscessus (Mabc) group, albeit clearly differentiated from Mabc. Compared with M. tuberculosis, a well-characterized human pathogen, the host innate immune response against Mmass infection is largely unknown. In this study, we show that Mmass robustly activates mRNA and protein expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in murine bone marrow-derived macrophages (BMDMs). Toll-like receptor (TLR)-2 and myeloid differentiation primary response gene 88 (MyD88), but neither TLR4 nor Dectin-1, are involved in Mmass-induced TNF-α or IL-6 production in BMDMs. Mmass infection also activates the mitogen-activated protein kinase (MAPKs; c-Jun N-terminal kinase (JNK), ERK1/2 and p38 MAPK) pathway. Mmass-induced TNF-α and IL-6 production was dependent on JNK activation, while they were unaffected by either the ERK1/2 or p38 pathway in BMDMs. Additionally, intracellular reactive oxygen species (ROS), NADPH oxidase-2, and nuclear factor-κB are required for Mmass-induced proinflammatory cytokine generation in macrophages. Furthermore, the S morphotype of Mmass showed lower overall induction of pro-inflammatory (TNF-α, IL-6, and IL-1β) and anti-inflammatory (IL-10) cytokines than the R morphotype, suggesting fewer immunogenic characteristics for this clinical strain. Together, these results suggest that Mmass-induced activation of host proinflammatory cytokines is mediated through TLR2-dependent JNK and ROS signaling pathways.

ABSTRACT Mycobacterium tuberculosis is a contagious agent that causes tuberculosis. A specific type (called the K cluster) of M. tuberculosis with 10 copies of IS 6110 in restriction fragment length polymorphism (RFLP) has been found in about 4% of M. tuberculosis isolates in Korea. Here, we report the complete genome sequence of M. tuberculosis Korean strain KIT87190 belonging to the K cluster.

With increasing international interchange of personnel, international monitoring is necessary to decrease tuberculosis incidence in the world. This study aims to develop a new tool to determine origin of Mycobacterium tuberculosis strains isolated from Filipino patients living in Korea. Thirty-two variable number tandem repeat (VNTR) loci were used for discrimination of 50 Filipino M. tuberculosis strains isolated in the Philippines, 317 Korean strains isolated in Korea, and 8 Filipino strains isolated in Korea. We found that the VNTR loci 0580, 0960, 2531, 2687, 2996, 0802, 2461, 2163a, 4052, 0424, 1955, 2074, 2347, 2401, 3171, 3690, 2372, 3232, and 4156 had different mode among copy numbers or exclusively distinct copy number in VNTR typing between Filipino and Korean M. tuberculosis strains. When these differences of the VNTR loci were applied to 8 Filipino M. tuberculosis strains isolated in Korea, 6 of them revealed Filipino type while 2 of them had Korean type. Using the differences of mode or repeated number of VNTR loci were very useful in distinguishing the Filipino strain from Korean strain.

BACKGROUND:Bacteria of the Mycobacterium abscessus group are the second most common pathogens responsible for lung disease caused by nontuberculous mycobacteria in Korea. There is still a lack of studies investigating the genetic mechanisms involved in M. abscessus resistance to antibiotics other than clarithromycin. This study investigated the characteristics of drug resistance exhibited by M. abscessus clinical isolates from Korea.METHODS:We performed drug susceptibility testing for a total of 404 M. abscessus clinical strains. Subspecies were differentiated by molecular biological methods and examined for mutations in drug resistance-related genes.RESULTS:Of the 404 strains examined, 202 (50.00%), 199 (49.26%), and 3 (0.74%) strains were identified as M. abscessus, M. massiliense, and M. bolletii, respectively. Of the 152 clarithromycin-resistant strains, 6 possessed rrl mutations, while 4 of the 30 amikacin-resistant strains contained rrs mutations, and 5 of the 114 quinolone-resistant strains had gyr mutations. All mutant strains had high minimal inhibitory concentration values for the antibiotics.CONCLUSIONS:Our results showed the distribution of the strains with mutations in drug resistance-related genes was low in the M. abscessus group. Furthermore, we performed drug susceptibility testing and sequence analyses to determine the characteristics of these genes in the M. abscessus group.

The PE (Pro-Glu) and PPE (Pro-Pro-Glu) multigene families are unique to mycobacteria, and are highly expanded in the pathogenic members of this genus. We determined the intra-subspecies genetic variability of the MACPPE12 gene, which is a specific PPE gene in Mycobacterium avium subsp. hominissuis (MAH), using 334 MAH isolates obtained from different isolation sources (222 human isolates, 145 Japanese and 77 Korean; 37 bathroom isolates; and 75 pig isolates). In total, 31 single-nucleotide polymorphisms (SNPs), which consisted of 16 synonymous SNPs and 15 nonsynonymous SNPs, were determined through comparison with the MACPPE12 gene sequence of MAH strain 104 as a reference. As the result, the 334 MAH isolates were classified into 19 and 13 different sequevars at the nucleic acid level (NA types) and amino acid level (AA types), respectively. Among the 13 AA types, only one type, the AA02 type, presented various NA types (7 different types) with synonymous SNPs, whereas all other AA types had a one-to-one correspondence with the NA types. This finding suggests that AA02 is a longer discernible lineage than the other AA types. Therefore, AA02 was classified as an ancestral type of the MACPPE12 gene, whereas the other AA types were classified as modern types. The ubiquitous presence of AA02 in all of the isolation sources and all different sequevars classified by the hsp65 genotype further supports this classification. In contrast to the ancestral type, the modern types showed remarkable differences in distribution between human isolates and pig isolates, and between Japanese isolates and Korean isolates. Divergence of the MACPPE12 gene may thus be a good indicator to characterize MAH strains in certain areas and/or hosts.

BACKGROUND:Variable-number tandem repeat (VNTR) typing is a promising method to discriminate the Mycobacterium tuberculosis isolates in molecular epidemiology. The purpose of this study is to determine the optimal VNTR combinations for discriminating isolated M. tuberculosis strains in Korea.METHODS:A total of 317 clinical isolates collected throughout Korea were genotyped by using the IS6110 restriction fragment length polymorphism (RFLP), and then analysed for the number of VNTR copies from 32 VNTR loci.RESULTS:THE RESULTS OF DISCRIMINATORY POWER ACCORDING TO DIVERSE COMBINATIONS WERE AS FOLLOWS: 25 clusters in 83 strains were yielded from the internationally standardized 15 VNTR loci (Hunter-Gaston discriminatory index [HGDI], 0.9958), 25 clusters in 65 strains by using IS6110 RFLP (HGDI, 0.9977), 14 clusters in 32 strains in 12 hyper-variable VNTR loci (HGDI, 0.9995), 6 clusters in 13 strains in 32 VNTR loci (HDGI, 0.9998), and 7 clusters in 14 strains of both the 12 hyper-variable VNTR and IS6110 RFLP (HDGI, 0.9999).CONCLUSION:The combination of 12 hyper-variable VNTR typing can be an effective tool for genotyping Korean M. tuberculosis isolates where the Beijing strains are predominant.

BACKGROUND:Notified tuberculosis (TB) cases in Korea have not decreased over the last decade (2001-2010).METHODS:To clarify the reasons, we analyzed an annual report on notified tuberculosis patients and age-specific population drift in Korea.RESULTS:Compared to the age-specific notified TB cases between 2001 and 2010, distinctive features in notified TB cases and new cases increased markedly in people aged 45-54 years and in patients over 65 years old, whereas those between 15-34 years in 2010 decreased drastically. In particular, notified TB individuals over 65 years old occupied 29.6% of the cases in 2010, which was 1.5 times higher than that in 2001. The main reason not to decrease in notified TB patients for the last decade (2001-2010) was due to the increasing elderly population as well as the aging of baby boomers, which have a higher risk of TB development.CONCLUSION:Korea needs to pay attention to the older population in order to successfully decrease the burden of TB in the future.

ABSTRACT Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine available against tuberculosis, and the strains used worldwide represent a family of daughter strains with distinct genotypic characteristics. Here, we report the complete genome sequence of M. bovis BCG Korea, the strain that will be actually used in Korea for vaccine production.

In order to characterize molecular mechanisms of first- and second-line drug resistance in Mycobacterium tuberculosis and to evaluate the use of molecular markers of resistance, we analyzed 62 multidrug-resistant, 100 extensively drug-resistant, and 30 pan-susceptible isolates from Korean tuberculosis patients. Twelve genome regions associated with drug resistance, including katG, ahpC, and inhA promoter for isoniazid (INH); embB for ethambutol (EMB), rpoB for rifampin (RIF), pncA for pyrazinamide (PZA), gyrA for fluoroquinolones; rpsL, gidB, and rrs for streptomycin; rrs and eis for kanamycin (KM); rrs and tylA for capreomycin (CAP); and rrs for amikacin (AMK) were amplified simultaneously by polymerase chain reaction, and the DNA sequences were determined. We found mutations in 140 of 160 INH-resistant isolates (87.5%), 159 of 162 RIF-resistant isolates (98.15%), 127 of 143 EMB-resistant isolates (88.8%), 108 of 123 ofloxacin-resistant isolates (87.8%), and 107 of 122 PZA-resistant isolates (87.7%); 43 of 51 STM-resistant isolates (84.3%), 15 of 17 KM-resistant isolates (88.2%), and 14 of 15 (AMK and CAP)-resistant isolates (93.3%) had mutations related to specific drug resistance. In addition, the sequence analyses of the study revealed many novel mutations involving these loci. This result suggests that mutations in the rpoB531, katGSer315Thr, and C-15T in the inhA promoter region, and gyrA94, embB306, pncA159, rpsL43, and A1401G in the rrs gene could serve as useful markers for rapid detection of resistance profile in the clinical isolates of M. tuberculosis in Korea, with potentials for the new therapeutic benefits in actual clinical practice.

Aminoglycosides are key drugs for the treatment of multidrug-resistant tuberculosis. A total of 97 extensively drug-resistant (XDR) and 29 pan-susceptible Mycobacterium tuberculosis isolates from Korean tuberculosis patients were analyzed to characterize mutations within the rrs, rpsL, gidB, eis and tlyA genes. Thirty (56.6 %) of the 53 streptomycin (STR)-resistant strains had a rpsL mutation and eight strains (15.1 %) had a rrs (514 or 908 site) mutation, whereas 11 (20.8 %) of the 53 STR-resistant strains had a gidB mutation without rpsL or either rrs mutation. Most of the gidB mutations conferred low-level STR resistance, and 22 of these mutations were novel. Mutation at position 1401 in rrs lead to resistance to kanamycin (80/95 = 84.2 %; KAN), amikacin (80/87 = 92.0 %; AMK), and capreomycin (74/86 = 86.0 %; CAP). In this study, 13.7 % (13/95) of KAN-resistant strains showed eis mutations, including 4 kinds of novel mutations. Isolates with eis structural gene mutations were cross-resistant to STR, KAN, CAP, and AMK. Here, 5.8 % (5/86) of the CAP-resistant strains harbored a tlyA mutation that included 3 different novel point mutations. Detection of the A1401G mutation appeared to be 100 % specific for the detection of resistance to KAN and AMK. These data establish the presence of phenotypic XDR strains using molecular profiling and are helpful to understanding of aminoglycoside resistance at the molecular level.

Many laboratories validate DST of the second-line drugs by BACTEC MGIT 960 system. The objective of this study is to evaluate the critical concentration and perform DST for the 2nd line drugs. We evaluated 193 clinical strains of M. tuberculosis isolated from patients in South Korea. Testing the critical concentration of six second-line drugs was performed by MGIT 960 and compared with L-J proportion method. The critical concentration was determined to establish the most one that gave the difference between drug resistance and susceptibility in MGIT960 system. Good agreement of the following concentrations was found: Concordance was 95% for 0.5  μ g/mL of moxifloxacin; 93.6%, 1.0  μ g/mL of levofloxacin; 97.5%, 2.5  μ g/mL of kanamycin; 90.6%, 2.5  μ g/mL of capreomycin; 86.2%, 5.0  μ g/mL of ethionamide; and 90.8%, 2.0  μ g/mL of ρ-aminosalicylic acid. The critical concentrations of the four drugs, moxifloxacin, levofloxacin, kanamycin, and capreomycin, were concordant and reliable for testing 2nd line drug resistance. Further study of ethionamide and ρ -aminosalicylic acid is required.

Mycobacterium abscessus (Mabc) is an emerging human pathogen. Less is known about the host immune response to Mabc than to M. tuberculosis. Here, we examined the intracellular signaling pathways that govern the expression of chemokines including (C-C motif) ligand 2 (CCL2) and (C-X-C motif) ligand 2 (CXCL2) in macrophages after infection with Mabc. Specifically, Mabc triggered the generation of reactive oxygen species (ROS) and the production of CCL2 and CXCL2 in murine bone marrow-derived macrophages (BMDMs). Mabc-induced CCL2, but not CXCL2, was dependent on the generation of ROS. Toll-like receptor (TLR) 2, MyD88, but not TRIF, was required for Mabc-induced CCL2 and CXCL2 expression. Additionally, Mabc infection significantly induced nuclear factor (NF)-κB nuclear translocation and luciferase activity. The activation of NF-κB was required for Mabc-induced CCL2, but not CXCL2 expression. Moreover, Mabc-induced ROS generation was required for NF-κB activation. Treatment of BMDMs with Mabc rapidly induced the activation of mitogen-activated protein kinase (MAPKs) pathways. Interestingly, CCL2 expression was dependent on the activation of JNK and ERK1/2 pathways, whereas it was negatively regulated by the p38 MAPK pathway. In contrast, Mabc-dependent CXCL2 expression was not regulated by MAPK pathways. These data suggest that intracellular ROS generation is required for innate and inflammatory responses during Mabc infection of macrophages.

The current standard of treatment against tuberculosis consists of a cocktail of first-line drugs, including isoniazid and pyrazinamide. Although these drugs are known to be bactericidal, contribution of host cell responses in the context of antimycobacterial chemotherapy, if any, remains unknown. We demonstrate that isoniazid and pyrazinamide promote autophagy activation and phagosomal maturation in Mycobacterium tuberculosis (Mtb)-infected host cells. Treatment of Mtb-infected macrophages with isoniazid or pyrazinamide caused significant activation of cellular and mitochondrial reactive oxygen species and autophagy, which was triggered by bacterial hydroxyl radical generation. Mycobacterium marinum-infected autophagy-defective, atg7 mutant Drosophila exhibited decreased survival rates, which could not be rescued by antimycobacterial treatment, indicating that autophagy is required for effective antimycobacterial drug action in vivo. Moreover, activation of autophagy by antibiotic treatment dampened Mtb-induced proinflammatory responses in macrophages. Together, these findings underscore the importance of host autophagy in orchestrating successful antimicrobial responses to mycobacteria during chemotherapy.

Identification of rapidly growing mycobacteria (RGM) is problematic because there are many taxonomic changes. 16S rRNA gene is commonly used to identify Mycobacterium species, but alternative gene targets have been introduced for more accurate identification. We report a rare case of a prosthetic knee infection due to Mycobacterium wolinskyi. The isolate was not identified by 16S rRNA gene sequencing alone and substantially confirmed by rpoB gene sequencing. The identification was delayed because our laboratory did not routinely identify RGM to the species level. Simultaneous sequencing of both 16S rRNA and rpoB genes will allow rapid and accurate identification of M. wolinskyi isolates.

Introduction The variable-number tandem repeats (VNTR) typing is a promising method to discriminate M. tuberculosis isolates in molecular epidemiology. Objectives The purpose of this study was to evaluate already known VNTR loci and to select best combination of VNTR loci set for discrimination Korean TB strains. Methods The 307 clinical isolates collected from throughout Korea were genotyped using IS 6110 RFLP, and analyzed the number of VNTR copies for the 32 VNTR loci including Supply-15, Supply-24 and JATA-15(except QUB-18); MIRU-02, -04, -10, -16, -20, -23, -24, -26, -27, -31, -39,- 40, ETR-A, -B, -C, QUB-11a, -11b, -15, -26, Mtub-04, -21, -24, -29, -30, -34, -39, VNTR-2372, -3232, -3336, -3820, -4120, -4156. And then, allelic diversity ( h ) and the Hunter-Gaston discriminatory index (HGDI) were calculated to evaluate discriminatory power of locus and combination of VNTR loci. Results As a results of analysis of the 32 VNTR loci, we found that 12 loci (MIRU-26, -31, QUB-11a, -11b, -26, Mtub-04, -21, VNTR-3232, -3336, -3820, -4120, -4156) showed high discrimination power (each h values of them was over 0.6). This new 12-locus combination for Korea TB strains was designated as a KIT-12. The discriminatory index (HGDI) of IS 6110 RFLP and VNTRs of Supply-15, Supply-24, JATA-15, and KIT-12 was 0.9992, 0.9980, 0.9987, 0.9992, and 0.9997 respectively. Also the percentage of clustered cases was 16.6%(51/307), 21.8%(67/307), 16.9%(52/307), 14.3%(44/307), 6.8%(21/307) respectively. Conclusions The newly proposed VNTR typing system, KIT-12 loci can be effective tool for M. tuberculosis genotyping in Korea where the Beijing strains are predominant.

The phenotypic resistance to ethambutol (EMB) in Mycobacterium tuberculosis with embB gene mutations is still unclear. This study was designed to better understand EMB resistance due to embB gene mutation. Sequencing analysis of the embB gene was performed for 124 EMB-susceptible and 93 EMB-resistant M. tuberculosis strains isolated from South Korea. The MIC was determined for EMB-susceptible M. tuberculosis strains with the embB mutation and wild-type on Löwenstein-Jenson (LJ) solid medium in duplicate. Two (2.8 %) of 72 pan-susceptible, two (9.1 %) of 22 any-drug-resistant but EMB-susceptible, nine (30.0 %) of 30 multidrug-resistant (MDR) but EMB-susceptible and 84 (90.3 %) of 93 EMB-resistant M. tuberculosis strains possessed embB mutations at various codons including 306, 319, 354, 360, 399, 405, 406, 459 and 497. Strains with embB mutations at codons 306, 354, 399, 405 and 497 had highly pronounced EMB resistance, while strains with mutations at codons 319 and 406 mutations were moderately resistant and those with an embB459 mutation were EMB-susceptible at the critical concentration (2.0 µg ml(-1)) on LJ solid medium. However, the mean MIC for strains with embB mutations (1.42 µg ml(-1)) was higher than that for strains without the embB mutation (1.0 µg ml(-1)) in EMB-susceptible M. tuberculosis isolates (P = 0.0052). Three novel embB mutations at codons 399, 405 and 459 were identified in this study. These results support the hypothesis that embB mutation except for a few specific mutation types may be the main cause of EMB resistance.

ABSTRACT We report the first case of Segniliparus rotundus pneumonia in an adult with non-cystic fibrosis bronchiectasis. All isolates were identified as S. rotundus by 16S rRNA gene sequencing and rpoB PCR-restriction analysis. Antibiotic therapy with clarithromycin and ciprofloxacin for 2 months improved the patient's condition and achieved successful sputum conversion.