Mycobacterium abscessus infection leads to enhanced production of type 1 interferon and NLRP3 inflammasome activation in murine macrophages via mitochondrial oxidative stress.

Mycobacterium abscessus (MAB) is a rapidly growing mycobacterium (RGM), and infections with this pathogen have been increasing worldwide. Recently, we reported that rough type (MAB-R) but not smooth type (MAB-S) strains enhanced type 1 interferon (IFN-I) secretion via bacterial phagosome escape, contributing to increased virulence. Here, we sought to investigate the role of mitochondrial oxidative stress in bacterial survival, IFN-I secretion and NLRP3 inflammasome activation in MAB-infected murine macrophages. We found that live but not heat-killed (HK) MAB-R strains increased mitochondrial ROS (mtROS) and increased release of oxidized mitochondrial DNA (mtDNA) into the cytosol of murine macrophages compared to the effects of live MAB-S strains, resulting in enhanced NLRP3 inflammasome-mediated IL-1ß and cGAS-STING-dependent IFN-I production. Treatment of the infected macrophages with mtROS-modulating agents such as mito-TEMPO or cyclosporin A reduced cytosolic oxidized mtDNA, which inhibited the MAB-R strain-induced production of IL-1ß and IFN-I. The reduced cytosolic oxidized mtDNA also inhibited intracellular growth of MAB-R strains via cytosolic escape following phagosomal rupture and via IFN-I-mediated cell-to-cell spreading. Moreover, our data showed that mtROS-dependent IFN-I production inhibited IL-1ß production, further contributing to MAB-R intracellular survival in murine macrophages. In conclusion, our data indicated that MAB-R strains enhanced IFN-I and IL-1ß production by inducing mtROS as a pathogen-associated molecular pattern (PAMP). These events also enhance bacterial survival in macrophages and dampen inflammation, which contribute to the pathogenesis of MAB-R strains.

A description of Mycobacterium chelonae subsp. gwanakae subsp. nov., a rapidly growing mycobacterium with a smooth colony phenotype due to glycopeptidolipids.

Three rapidly growing mycobacterial strains, MOTTH4W, MOTT36WT and MOTT68W, were isolated from the sputa of three independent Korean patients co-infected with Mycobacterium yongonense Type II strains. The 16S rRNA gene sequences of all three strains were unique, which were closest to that of Mycobacterium chelonae subsp. bovis KCTC 39630T (99.9 % similarity). Multilocus sequence typing analysis targeting 10 housekeeping genes including hsp65 and rpoB revealed the distinct phylogenetic location of these strains, which were clustered with M. chelonae subsp. chelonae ATCC 35752T and M. chelonae subsp. bovis KCTC 39630T. Phylogenetic analysis based on whole genome sequences revealed a 95.89 % average nucleotide identity (ANI) value with M. chelonae subsp. chelonae, slightly higher than the 95.0 % ANI criterion for determining a novel species. In addition, phenotypic characteristics such as a smooth colony morphology and growth inhibition at 37 °C, distinct MALDI-TOF MS profiles of extracted total lipids due to surface glycopeptidolipids, and distinct drug susceptibility profiles further supported the taxonomic characterization of these strains as representing a novel subspecies of Mycobacterium chelonae. Mycobacterium chelonae subsp. gwanakae subsp. nov. is proposed and the type strain is MOTT36WT (=KCTC 29127T=JCM 32454T).

Description of Mycobacterium chelonae subsp. bovis subsp. nov., isolated from cattle (Bos taurus coreanae), emended description of Mycobacterium chelonae and creation of Mycobacterium chelonae subsp. chelonae subsp. nov.

Three rapidly growing mycobacterial strains, QIA-37T, QIA-40 and QIA-41, were isolated from the lymph nodes of three separate Korean native cattle, Hanwoo (Bos taurus coreanae). These strains were previously shown to be phylogenetically distinct but closely related to Mycobacterium chelonae ATCC 35752T by taxonomic approaches targeting three genes (16S rRNA, hsp6 and rpoB) and were further characterized using a polyphasic approach in this study. The 16S rRNA gene sequences of all three strains showed 99.7 % sequence similarity with that of the M. chelonae type strain. A multilocus sequence typing analysis targeting 10 housekeeping genes, including hsp65 and rpoB, revealed a phylogenetic cluster of these strains with M. chelonae. DNA-DNA hybridization values of 78.2 % between QIA-37T and M. chelonae indicated that it belongs to M. chelonae but is a novel subspecies distinct from M. chelonae. Phylogenetic analysis based on whole-genome sequences revealed a 95.44±0.06 % average nucleotide identity (ANI) value with M. chelonae, slightly higher than the 95.0 % ANI criterion for determining a novel species. In addition, distinct phenotypic characteristics such as positive growth at 37 °C, at which temperature M. chelonae does not grow, further support the taxonomic status of these strains as representatives of a novel subspecies of M. chelonae. Therefore, we propose an emended description of Mycobacterium chelonae, and descriptions of M. chelonae subsp. chelonae subsp. nov. and M. chelonae subsp. bovis subsp. nov. are presented; strains ATCC 35752T(=CCUG 47445T=CIP 104535T=DSM 43804T=JCM 6388T=NCTC 946T) and QIA-37T (=KCTC 39630T=JCM 30986T) are the type strains of the two novel subspecies.

Mycobacterium paraintracellulare sp. nov., for the genotype INT-1 of Mycobacterium intracellulare.

Three mycobacterial strains, isolated from independent Korean patients with pulmonary infections, belonging to the Mycobacterium intracellulare genotype 1 (INT-1) were characterized using a polyphasic approach. The sequences of the 16S rRNA gene and internal transcribed spacer 1 (ITS1) of the INT-1 strains were identical to those of Mycobacterium intracellulare ATCC 13950T. However, multilocus sequence typing (MLST) analysis targeting five housekeeping genes (hsp65, rpoB, argG, gnd and pgm) revealed the phylogenetic separation of these strains from M. intracellulare ATCC 13950T. DNA-DNA hybridization values of >70 % confirmed that the three isolates belong to the same species, while the values of <70 % between one of them and the type strains of M. intracellulare and Mycobacterium chimaera confirmed their belonging to a distinct species. In addition, phenotypic characteristics such as positive growth on MacConkey agar and in acidic broth culture, unique matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS profiles of lipids, and unique mycolic acids profiles further supported the taxonomic status of these strains as representatives of a novel species of the Mycobacterium avium complex named Mycobacterium paraintracellulare. The type strain is MOTT64T (=KCTC 29084T=JCM 30622T).

Identification of ISMyo2, a novel insertion sequence element of IS21 family and its diagnostic potential for detection of Mycobacterium yongonense.

BACKGROUND: Mycobacterium yongonense, as a novel member of the M. avium complex (MAC), was recently reported to be isolated from human specimens in South Korea and Italy. Due to its close relatedness to other MAC members, particularly M. intracellulare in taxonomic aspects, the development of a novel diagnostic method for its specific detection is necessary for clinical or epidemiologic purposes. METHODS: Using the Mycobacterium yongonense genome information, we have identified a novel IS-element, ISMyo2. Targeting the ISMyo2 sequence, we developed a real-time PCR method and applied the technique to Mycobacterial genomic DNA. RESULTS: To identify proper nucleic acid targets for the diagnosis, comparisons of all insertion sequence (IS) elements of 3 M. intracellulare and 3 M. yongonense strains, whose complete genome sequences we reported recently, led to the selection of a novel target gene, the M. yongonense-specific IS element, ISMyo2 (2,387 bp), belonging to the IS21 family. Next, we developed a real-time PCR method using SYBR green I for M. yongonense-specific detection targeting ISMyo2, producing a 338-bp amplicon. When this assay was applied to 28 Mycobacterium reference strains and 63 MAC clinical isolates, it produced amplicons in only the 6 M. yongonense strains, showing a sensitivity of 100 fg of genomic DNA, suggesting its feasibility as a diagnostic method for M. yongonense strains. CONCLUSIONS: We identified a novel ISMyo2 IS element belonging to the IS21 family specific to M. yongonense strains via genome analysis, and a real-time PCR method based on its sequences was developed.

Development of a peptide nucleic acid-based multiprobe real-time PCR method targeting the hsp65 gene for differentiation among Mycobacterium abscessus strains.

Recently, the need to distinguish between members of the Mycobacterium abscessus group has gained increasing attention. Here, we introduced a novel peptide nucleic acid (PNA) real-time PCR method targeting the hsp65 gene in order to distinguish between four subspecies within the M. abscessus group (M. abscessus and 3 types of M. massiliense).

Mycobacterium anyangense sp. nov., a rapidly growing species isolated from blood of Korean native cattle, Hanwoo (Bos taurus coreanae).

From the whole blood of Korean native cattle, Hanwoo (Bos taurus coreanae), a previously undescribed, rapidly growing, scotochromogenic isolate of the genus Mycobacterium is reported. Its 16S rRNA gene sequence, and the sequences of three other genes (hsp65, recA and rpoB) were unique and phylogenetic analysis based on 16S rRNA gene sequence (1420 bp) placed the organism into the rapidly growing Mycobacterium group close to Mycobacterium smegmatis (98.5% sequence similarity). However, phylogenetic analyses based on three different gene sequences (hsp65, recA and rpoB) revealed its location to be distinct from the branch of rapidly growing species. Culture and biochemical characteristics were generally similar to those of Mycobacterium fortuitum. Unique matrix-assisted laser desorption ionization time-of-flight (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 representative of a novel species of rapidly growing mycobacteria named Mycobacterium anyangense. The type strain is strain QIA-38(T) ( = JCM 30275(T) = KCTC 29443(T)).

Separation of Mycobacterium abscessus into subspecies or genotype level by direct application of peptide nucleic acid multi-probe- real-time PCR method into sputa samples.

BACKGROUND: 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. METHODS: 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. RESULTS: 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]. CONCLUSIONS: 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.

Mycobacterium paragordonae sp. nov., a slowly growing, scotochromogenic species closely related to Mycobacterium gordonae.

A previously undescribed, slowly growing, scotochromogenic mycobacterial strain (49061(T)) was isolated from a patient with pulmonary infections during the hsp65-sequence-based identification of Korean clinical isolates. Its 16S rRNA gene sequence was unique and the phylogenetic analysis based on 16S rRNA gene sequence (1393 bp) placed the organism into the slow-growing Mycobacterium group close to Mycobacterium gordonae (99.0 % sequence similarity). Growth characteristics and acid-fastness also 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 49061(T) was 25-30 °C, and it was not able to grow at 37 °C on 7H10 agar slants. Unique MALDI-TOF MS profiles of lipids, phylogenetic analysis based on another two 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 representative of a distinct species from M. gordonae. It was concluded that the strain represents a novel species for which the name Mycobacterium paragordonae is proposed with the type strain 49061(T) ( = JCM 18565(T) = KCTC 29126(T)).

Rough colony morphology of Mycobacterium massiliense Type II genotype is due to the deletion of glycopeptidolipid locus within its genome.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Mycobacterium yongonense sp. nov., a slow-growing non-chromogenic species closely related to Mycobacterium intracellulare.

A slow-growing non-chromogenic mycobacterium was isolated from a patient with pulmonary disease. Phenotypically, strain 05-1390(T) was similar to Mycobacterium intracellulare ATCC 13950(T). The 16S rRNA gene sequence (1385 bp) of strain 05-1390(T) showed a high degree of similarity to those of the M. intracellulare complex, namely Mycobacterium marseillense 5351974(T) (100 %), M. intracellulare ATCC 13950(T) (99.8 %) and Mycobacterium chimaera DSM 44623(T) (99.9 %). Phylogenetic analysis based on internal transcribed spacer 1 (ITS1) and the hsp65 gene indicated that strain 05-1390(T) was closely related to M. intracellulare ATCC 13950(T), but that it was a distinct phylogenetic entity. Of particular interest, an analysis based on the rpoB gene (701 bp) showed that it is closely related to Mycobacterium parascrofulaceum ATCC BAA-614(T) (99.4 %), a scotochromogenic strain, rather than to the M. intracellulare-related strains. Unique MALDI-TOF MS profiles also supported the taxonomic status of this strain as a distinct species. These data support the conclusion that strain 05-1390(T) represents a novel mycobacterial species, for which the name Mycobacterium yongonense sp. nov. is proposed; the type strain is 05-1390(T) ( = DSM 45126(T) = KCTC 19555(T)).

Mycobacterium parakoreense sp. nov., a slowly growing non-chromogenic species related to Mycobacterium koreense, isolated from a human clinical specimen.

A previously undescribed, slowly growing, non-chromogenic Mycobacterium strain (299(T)) was isolated from the sputum sample of a patient with a symptomatic pulmonary infection. Phenotypically, strain 299(T) was generally similar to Mycobacterium koreense DSM 45576(T) and Mycobacterium triviale ATCC 23292(T). The 16S rRNA gene sequence of strain 299(T) was similar to that of M. koreense DSM 45576(T) (GenBank accession no. AY734996, 99.5% similarity); however, it differed substantially from that of M. triviale ATCC 23292(T) (X88924, 98.2%). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 299(T) clustered together with M. koreense DSM 45576(T) and M. triviale ATCC 23292(T), supported by high bootstrapping values (99%). Unique mycolic acid profiles and phylogenetic analysis based on two different chronometer molecules, 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 299(T) represents a novel mycobacterial species, for which the name Mycobacterium parakoreense sp. nov. is proposed. The type strain is 299(T) (=DSM 45575(T)=KCTC 19818(T)).

Potential Use of Mycobacterium paragordonae for Antimycobacterial Drug Screening Systems.

Our recent genome-based study indicated that Mycobacterium paragordonae (Mpg) has evolved to become more adapted to an intracellular lifestyle within free-living environmental amoeba and its enhanced intracellular survival within Acanthamoeba castellanii was also proved. Here, we sought to investigate potential use of Mpg for antimycobacterial drug screening systems. Our data showed that Mpg is more susceptible to various antibiotics compared to the close species M. marinum (Mmar) and M. gordonae, further supporting its intracellular lifestyle in environments, which would explain its protection from environmental insults. In addition, we developed two bacterial whole-cell-based drug screening systems using a recombinant Mpg stain harboring a luciferase reporter vector (rMpg-LuxG13): one for direct application to rMpg-LuxG13 and the other for drug screening via the interaction of rMpg-LuxG13 with A. castellanii. Direct application to rMpg-LuxG13 showed lower inhibitory concentration 50 (IC50) values of rifampin, isoniazid, clarithromycin, and ciprofloxacin against Mpg compared to Mmar. Application of drug screening system via the interaction of rMpg-LuxG13 with A. castellanii also exhibited lower IC50 values for rifampin against Mpg compared to Mmar. In conclusion, our data indicate that Mpg is more susceptible to various antibiotics than other strains. In addition, our data also demonstrate the feasibility of two whole cell-based drug screening systems using rMpg-LuxG13 strain for the discovery of novel anti-mycobacterial drugs.

Protection against tuberculosis achieved by dissolving microneedle patches loaded with live Mycobacterium paragordonae in a BCG prime-boost strategy.

Introduction: Skin vaccination using dissolving microneedle patch (MNP) technology for transdermal delivery is a promising vaccine delivery strategy to overcome the limitations of the existing vaccine administration strategies using syringes. To improve the traditional microneedle mold fabrication technique, we introduced droplet extension (DEN) to reduce drug loss. Tuberculosis remains a major public health problem worldwide, and BCG revaccination had failed to increase the protective efficacy against tuberculosis. We developed an MNP with live Mycobacterium paragordonae (Mpg) (Mpg-MNP) as a candidate of tuberculosis booster vaccine in a heterologous prime-boost strategy to increase the BCG vaccine efficacy. Materials and methods: The MNPs were fabricated by the DEN method on a polyvinyl alcohol mask film and hydrocolloid-adhesive sheet with microneedles composed of a mixture of mycobacteria and hyaluronic acid. We assessed the transdermal delivery efficiency by comparing the activation of the dermal immune system with that of subcutaneous injection. A BCG prime Mpg-MNP boost regimen was administered to a mouse model to evaluate the protective efficacy against M. tuberculosis. Results: We demonstrated the successful transdermal delivery achieved by Mpg-MNP compared with that observed with BCG-MNP or subcutaneous vaccination via an increased abundance of MHCII-expressing Langerin+ cells within the dermis that could migrate into draining lymph nodes to induce T-cell activation. In a BCG prime-boost regimen, Mpg-MNP was more protective than BCG-only immunization or BCG-MNP boost, resulting in a lower bacterial burden in the lungs of mice infected with virulent M. tuberculosis. Mpg-MNP-boosted mice showed higher serum levels of IgG than BCG-MNP-boosted mice. Furthermore, Ag85B-specific T-cells were activated after BCG priming and Mpg-MNP boost, indicating increased production of Th1-related cytokines in response to M. tuberculosis challenge, which is correlated with enhanced protective efficacy. Discussion: The MNP fabricated by the DEN method maintained the viability of Mpg and achieved effective release in the dermis. Our data demonstrate a potential application of Mpg-MNP as a booster vaccine to enhance the efficacy of BCG vaccination against M. tuberculosis. This study produced the first MNP loaded with nontuberculous mycobacteria (NTM) to be used as a heterologous booster vaccine with verified protective efficacy against M. tuberculosis.

Complete genome sequence of Mycobacterium intracellulare clinical strain MOTT-36Y, belonging to the INT5 genotype.

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.

Complete genome sequence of Mycobacterium intracellulare strain ATCC 13950(T).

Here we report the first complete genome sequence of Mycobacterium intracellulare ATCC 13950(T), a Mycobacterium avium complex (MAC) strain. This genome sequence will serve as a valuable reference for understanding the epidemiologic, biological, and pathogenic aspects of the disparity between MAC members.

Complete genome sequence of Mycobacterium intracellulare clinical strain MOTT-02.

Here, we report the first complete genome sequence of the Mycobacterium intracellulare clinical strain MOTT-02, which was previously grouped in the INT2 genotype of M. intracellulare. This genome sequence will serve as a valuable reference for improving the understanding of the disparity in the virulence and epidemiologic traits between M. intracellulare genotypes.

Complete genome sequence of Mycobacterium intracellulare clinical strain MOTT-64, belonging to the INT1 genotype.

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.

Mycobacterium koreense sp. nov., a slowly growing non-chromogenic species closely related to Mycobacterium triviale.

A novel slow-growing, non-chromogenic mycobacterium (strain 01-305(T)) was isolated from a patient with pulmonary dysfunction. Growth characteristics, acid-fastness and the results of 16S rRNA gene sequencing supported the placement of this strain within the genus Mycobacterium. Phenotypically, strain 01-305(T) was generally similar to Mycobacterium triviale ATCC 23292(T), but some unique biochemical characteristics were observed. The 16S rRNA gene sequence of strain 01-305(T) was similar to those of M. triviale ATCC 23290 (GenBank accession no. AY734996, 99.9 % similarity) and M. triviale ATCC 23291 (AY734995, 99.9 %); however, it differed substantially from that of M. triviale ATCC 23292(T) (X88924, 98.2 %). Phylogenetic analysis based on 16S rRNA gene sequences placed strain 01-305(T) in the slow-growing Mycobacterium group close to M. triviale ATCC 23290 and M. triviale ATCC 23291, but not M. triviale ATCC 23292(T). 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 01-305(T) represents a novel mycobacterial species, for which the name Mycobacterium koreense sp. nov. is proposed. The type strain is 01-305(T) ( = DSM 45576(T) = KCTC 19819(T)).

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.