Mycobacterium kiyosense sp. nov., a scotochromogenic slow-glowing species isolated from respiratory specimens.

Scotochromogenic slow-growing mycobacteria were isolated from the sputum or bronchoalveolar lavage fluid of 12 patients in Japan. From a comparison of the whole-genome sequences, the representative strain IWGMT90018-18076T and the unknown strains obtained from the patients were found to represent a novel species related to the Mycobacterium gordonae complex. The average nucleotide identity values of IWGMT90018-18076T with Mycobacterium vicinigordonae, Mycobacterium paragordonae and M. gordonae were 86.7, 82.5 and 82.2 %, respectively. The genome size of the representative strain IWGMT90018-18076T was approximately 6.3 Mbp, and the genomic DNA G+C content was 67.1 %. The major fatty acid methyl esters were C16 : 0 (37.71 %), C18 : 1ω9c (29.5 %) and C16 : 1ω7c (10.32 %). In this study, we performed phylogenetic analyses, physiological and biochemical characteristic tests, drug susceptibility tests and fatty acid profiling of the clinical isolates. On the basis of the results obtained, we propose that the unknown clinical isolates represent a novel species, 'Mycobacterium kiyosense sp. nov,' with the type strain being IWGMT90018-18076T (=JCM 34837T =KCTC 49725T).

Complete Chromosomal Genome Sequence of Mycobacterium sp. Strain IWGMT90018-18076.

We have isolated a strain that we believe is identical to strain IWGMT90018-an unidentified nontuberculous mycobacterium (NTM) species published in 1981-and named it IWGMT90018-18076. Here, we report its complete chromosomal genome sequence. This study will help us understand the diversity and pathogenicity of NTM.

Mycobacterium shigaense sp. nov., a slow-growing, scotochromogenic species, is a member of the Mycobacterium simiae complex.

Among non-tuberculous mycobacteria (NTM), the Mycobacterium simiae complex is one of the largest groups, consisting of 18 species of slow-growing mycobacteria. In 2009, a case of NTM-associated infectious skin disease was reported in Shiga Prefecture, Japan. The patient presented with scattered nodules on the chest, back and extremities, and an M. simiae-like organism was isolated from skin biopsy specimens obtained from one of these lesions. Based on several assessments, including multiple-gene analyses, biochemical characterization and drug susceptibility testing, we concluded that this isolate represented a novel species of NTM, and proposed the name 'Mycobacterium shigaense'. Since 2009, five more cases of NTM-associated infectious disease in which there was a suspected involvement of 'M. shigaense' have been reported. Interestingly, four of these six cases occurred in Shiga Prefecture. Here we performed multiple-gene phylogenetic analyses, physiological and biochemical characterization tests, drug susceptibility tests, and profiling of proteins, fatty acids and mycolic acids of eight clinical isolates from the six suspected 'M. shigaense' cases. The results confirmed that all of the clinical isolates were 'M. shigaense', a slow-growing, scotochromogenic species. Here M. shigaense is validly proposed as a new member of the M. simiae complex, with the type strain being UN-152T (=JCM 32072T=DSM 46748T).

Complete Genome Sequence of Mycobacterium shigaense.

Mycobacterium shigaense is a slowly growing scotochromogenic species and a member of the Mycobacterium simiae complex group. Here, we report the complete sequence of its genome, comprising a 5.2-Mb chromosome. The sequence will represent the essential data for future phylogenetic and comparative genome studies of the Mycobacterium simiae complex group.

Naturally occurring a loss of a giant plasmid from Mycobacterium ulcerans subsp. shinshuense makes it non-pathogenic.

Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU), a WHO-defined neglected tropical disease. All Japanese BU causative isolates have shown distinct differences from the prototype and are categorized as M. ulcerans subspecies shinshuense. During repeated sub-culture, we found that some M. shinshuense colonies were non-pigmented whereas others were pigmented. Whole genome sequence analysis revealed that non-pigmented colonies did not harbor a giant plasmid, which encodes elements needed for mycolactone toxin biosynthesis. Moreover, mycolactone was not detected in sterile filtrates of non-pigmented colonies. Mice inoculated with suspensions of pigmented colonies died within 5 weeks whereas those infected with suspensions of non-pigmented colonies had significantly prolonged survival (>8 weeks). This study suggests that mycolactone is a critical M. shinshuense virulence factor and that the lack of a mycolactone-producing giant plasmid makes the strain non-pathogenic. We made an avirulent mycolactone-deletion mutant strain directly from the virulent original.

Complete Genome Sequence of Mycobacterium ulcerans subsp. shinshuense.

Mycobacterium ulcerans subsp. shinshuense produces mycolactone and causes Buruli ulcer. Here, we report the complete sequence of its genome, which comprises a 5.9-Mb chromosome and a 166-kb plasmid (pShT-P). The sequence will represent the essential data for future phylogenetic and comparative genome studies of mycolactone-producing mycobacteria.

Clinical and microbiological features of definite Mycobacterium gordonae pulmonary disease: the establishment of diagnostic criteria for low-virulence mycobacteria.

BACKGROUND: Although Mycobacterium gordonae isolation from respiratory samples is usually regarded as contamination, M. gordonae can cause definite pulmonary disease. The establishment of a standard diagnostic criteria of pulmonary disease that is caused by this low virulence mycobacterium is obviously necessary. METHODS: We performed clinical research on over 200 cases in which M. gordonae was isolated over an 8-year period, focusing on the M. gordonae subtype. Sequence analysis of rpoB was performed to identify the genotypes. RESULTS: A total of 287 respiratory samples (209 cases) were positive for M. gordonae. Twenty-seven cases (12.9%) had a positive culture more than twice and 11 of these cases (5.3%) had more than three positive cultures. Ultimately, three cases (1.4%) were newly diagnosed as M. gordonae pulmonary disease using our own diagnostic criteria. In all of the identified M. gordonae cases, the cultures tested positive with a Mycobacteria Growth Indicator Tube test at 24 days; however, in patients with definitive pulmonary disease, the cultures were positive at 9 days. A subtype analysis revealed that all of the definitive disease cases had subtype C. CONCLUSION: The time taken to detect a positive culture and subtype of the isolates could be used as the diagnostic criteria for definite M. gordonae pulmonary disease.

Disseminated Mycobacterium gordonae and Mycobacterium mantenii infection with elevated anti-IFN-γ neutralizing autoantibodies.

A case of disseminated nontuberculous mycobacteria(l) (NTM) infection in a patient with positive neutralizing anti-interferon-γ (IFN-γ) autoantibodies involving bone, bronchus, systemic lymph nodes, and skin is reported. The causative NTMs were two different strains: Mycobacterium gordonae, which rarely causes true disease, and Mycobacterium mantenii, which is extremely rare. Anti-mycobacterial treatment successfully ameliorated all disseminated lesions. Although the concentration of anti-IFN-γ autoantibodies increased during the pre-treatment period, it gradually decreased after anti-mycobacterial treatment was started.

Complete genome sequence and comparative genomic analysis of Mycobacterium massiliense JCM 15300 in the Mycobacterium abscessus group reveal a conserved genomic island MmGI-1 related to putative lipid metabolism.

Mycobacterium abscessus group subsp., such as M. massiliense, M. abscessus sensu stricto and M. bolletii, are an environmental organism found in soil, water and other ecological niches, and have been isolated from respiratory tract infection, skin and soft tissue infection, postoperative infection of cosmetic surgery. To determine the unique genetic feature of M. massiliense, we sequenced the complete genome of M. massiliense type strain JCM 15300 (corresponding to CCUG 48898). Comparative genomic analysis was performed among Mycobacterium spp. and among M. abscessus group subspp., showing that additional ß-oxidation-related genes and, notably, the mammalian cell entry (mce) operon were located on a genomic island, M. massiliense Genomic Island 1 (MmGI-1), in M. massiliense. In addition, putative anaerobic respiration system-related genes and additional mycolic acid cyclopropane synthetase-related genes were found uniquely in M. massiliense. Japanese isolates of M. massiliense also frequently possess the MmGI-1 (14/44, approximately 32%) and three unique conserved regions (26/44; approximately 60%, 34/44; approximately 77% and 40/44; approximately 91%), as well as isolates of other countries (Malaysia, France, United Kingdom and United States). The well-conserved genomic island MmGI-1 may play an important role in high growth potential with additional lipid metabolism, extra factors for survival in the environment or synthesis of complex membrane-associated lipids. ORFs on MmGI-1 showed similarities to ORFs of phylogenetically distant M. avium complex (MAC), suggesting that horizontal gene transfer or genetic recombination events might have occurred within MmGI-1 among M. massiliense and MAC.

In vitro susceptibility of Mycobacterium tuberculosis isolates to an oral carbapenem alone or in combination with ß-lactamase inhibitors.

We evaluated the antituberculosis (anti-TB) activity of five ß-lactams alone or in combination with ß-lactamase inhibitors against 41 clinical isolates of Mycobacterium tuberculosis, including multidrug-resistant and extensively drug-resistant strains. Of those, tebipenem, an oral carbapenem, showed the most potent anti-TB activity against clinical isolates, with a MIC range of 0.125 to 8 µg/ml, which is achievable in the human blood. More importantly, in the presence of clavulanate, MIC values of tebipenem declined to 2 µg/ml or less.

Comparative evaluation of three immunochromatographic identification tests for culture confirmation of Mycobacterium tuberculosis complex.

BACKGROUND: The rapid identification of acid-fast bacilli recovered from patient specimens as Mycobacterium tuberculosis complex (MTC) is critically important for accurate diagnosis and treatment. A thin-layer immunochromatographic (TLC) assay using anti-MPB64 or anti-MPT64 monoclonal antibodies was developed to discriminate between MTC and non-tuberculosis mycobacteria (NTM). Capilia TB-Neo, which is the improved version of Capilia TB, is recently developed and needs to be evaluated. METHODS: Capilia TB-Neo was evaluated by using reference strains including 96 Mycobacterium species (4 MTC and 92 NTM) and 3 other bacterial genera, and clinical isolates (500 MTC and 90 NTM isolates). M. tuberculosis isolates tested negative by Capilia TB-Neo were sequenced for mpt64 gene. RESULTS: Capilia TB-Neo showed 100% agreement to a subset of reference strains. Non-specific reaction to M. marinum was not observed. The sensitivity and specificity of Capilia TB-Neo to the clinical isolates were 99.4% (99.6% for M. tuberculosis, excluding M. bovis BCG) for clinical MTC isolates and 100% for NTM isolates tested, respectively. Two M. tuberculosis isolates tested negative by Capilia TB-Neo: one harbored a 63-bp deletion in the mpt64 gene and the other possessed a 3,659-bp deletion from Rv1977 to Rv1981c, a region including the entire mpt64 gene. CONCLUSIONS: Capilia TB-Neo is a simple, rapid and highly sensitive test for identifying MTC, and showed better specificity than Capilia TB. However, Capilia TB-Neo still showed false-negative results with mpt64 mutations. The limitation should be recognized for clinical use.

[The outbreak of nosocomial infection by Mycobacterium chelonae chemovar niacinogenes and the cause of its spread].

Mycobacteria consist of 2 large groups: one is the tuberculosis complex, and the other is nontuberculous mycobacterium (NTM). Most of the NTM are generally non-virulent bacteria, but some NTMs have pathogenicity to humans. There are many reports of nosocomial infection cases caused by common bacteria such as multidrug-resistant Pseudomonas aeruginosa. Also, some cases of in-hospital infection due to NTM were reported. Unlike common bacteria, detection of mycobacteria is affected by various factors, such as stainability, time for colony forming, temperature and nutrition Mycobacterium chelonae chemovar niacinogenes was isolated from 5 patients in 73 nosocomial infection cases (60 patients and 13 suspected cases) at a certain hospital during the period from March 2007 until January 2009. One of the reasons for the expansion of infection and difficulty in identification of the bacteria was the properties of this mycobacterium. This bacterium was very faintly stained with Gram-staining. Therefore, this mycobacterium could only be detected at a hospital when Ziehl-Neelsen stain and the cultivation at 28 degrees C for more than 5 days were performed. MICs for Cefmenoxime and Tosufloxacin of the isolates were more than 128 microg/mL. The isolates and type strasin of M. chelonae chemovar niacinogenes were also resistant to other drugs.

Synthesis and structure of two new mycolactones isolated from M. ulcerans subsp. shinshuense.

Two new mycolactones, mycolactones S1 and S2, were isolated from culture agar of Mycobacterium ulcerans subsp. shinshuense. Their structures were established in a three-step procedure: (1) probable structures were speculated from MS analysis; (2) candidates were synthesized; (3) HPLC profiles were established for identification of the natural products. Newly isolated mycolactones correspond to the "oxidized forms" of mycolactone A/B, the causative toxin of Buruli ulcer, isolated from Mycobacterium ulcerans.

Congenital cystic adenomatoid malformation in adulthood complicated by Mycobacterium celatum infection.

A 25-year-old woman with no underlying disease presented with a large fluid-filled cavitary lesion in the right lung. Mycobacterium celatum was isolated from the cavitary fluid, and treatment with ethambutol, rifampicin, and clarithromycin was initiated. After 4 months of treatment, right lower pulmonary lobectomy was performed. Histological examination confirmed M. celatum infection as well as concurrent congenital cystic adenomatoid malformation (CCAM). M. celatum has been implicated in opportunistic infections. This infection, however, was related to underlying CCAM, which resulted in a large cavitary lesion. CCAM diagnosed in adulthood is rare, and is made more challenging by an infectious complication.

The evaluation of an identification algorithm for Mycobacterium species using the 16S rRNA coding gene and rpoB.

BACKGROUND: Conventional biochemical tests are the standard for the identification of Mycobacterium species, but molecular identifications are becoming more prevalent. The rpoB gene encodes the ß-subunit of RNA polymerase and is utilized for the identification of Mycobacterium species. In the present study, a stepwise Mycobacterium species identification algorithm using the 16S rRNA encoding gene and rpoB analysis was evaluated for its effectiveness. METHODS: A total of 172 clinical Mycobacterium isolates were tested, and concordant results were obtained with 108 strains by using the conventional method and molecular methods (AccuProbe or DDH method). RESULTS: In these 108 strains, 4 strains were identified by 16S rRNA gene analysis, but rpoB indicated no identical Mycobacterium species with more than 99% similarity. The remaining 64 strains were not identified by conventional method and commercial kits. Forty-two showed concordant results with 16S rRNA and rpoB analysis, and 13 strains were identified by 16S rRNA gene analysis although rpoB indicated no identical Mycobacterium species. On the other hand, 4 strains included 2 strains of Gordona and 2 strains of M. celatum type II which were identified by rpoB but not by 16S rRNA gene analysis. Finally, 5 strains could not be identified by analysis of either gene. The rpoB analysis can differentiate M. kansasii from M. gastri; M. malmoense from M. szulgai; M. abscessus from M. chelonae; M. peregrinum from M. septicum; M. porcinum from M. fortuitum; and M. farucinogense from M. senegalense-pairs that are not differentiated by 16S rRNA analysis. Additionally, Nocardia asteroids, Rhodococcus equi, Gordona aichiense, G. aurantiaca, G. bronchialis and G. terrae are able to be analyzed by using rpoB. CONCLUSIONS: The 16S rRNA gene identification is a rapid and prevalent method but still has some limitations. Therefore, the stepwise combination of rpoB with 16S rRNA gene analysis is an effective system for the identification of Mycobacterium species.

Mycobacterium heckeshornense lung infection that was diagnosed as Mycobacterium xenopi disease by DNA-DNA hybridization (DDH).

The DNA sequencing analyses of the 16S rRNA gene, rpoB and hsp65 were conducted to characterize six strains that had been identified as Mycobacterium xenopi by DNA-DNA hybridization (DDH) for past ten years in our hospital. The results revealed Mycobacterium heckeshornense infection in one of the six cases. A 47-year-old man, who had been treated for pneumonia, had pulmonary nontuberculous mycobacterial disease. The sputa from the patient were culture positive for mycobacterium in three times. And it was diagnosed as M. xenopiby DDH method. Chest X-ray showed fibrocavitary lesion in right upper lobe was successfully treated with clarithromycin for four weeks.

Immunopathological characteristics of immune reconstitution inflammatory syndrome caused by Mycobacterium parascrofulaceum infection in a patient with AIDS.

Immune reconstitution inflammatory syndrome (IRIS) caused by mycobacterium in patients with AIDS is often experienced in clinical practice. There is, however, a paucity of data documenting the histopathological findings and the pathogenesis. We determined the immunopathological characteristics of IRIS associated with Mycobacterium parascrofulaceum infection in an AIDS patient. A patient presented with pulmonary lymphadenitis and involvement of the pulmonary lingular segment. Portions of the involved lymph nodes and lung were excised, and the immunological properties were analyzed by immunohistochemical assays. The histological characteristics of lymph nodes showed a caseous necrosis. Histopathologically, the pulmonary lesion was composed of exudative and proliferative lesions. CD4(+), CD8(+), CD57(+), and CD25(+)/FoxP3(+) cells were observed in both types of lesions. Clusters of CD20(+) cells and GATA3(+) cells were predominantly observed in exudative lesions, while T-bet(+) cells were dominant in proliferative lesions. ROR-γ(+) cells were also observed in exudative lesions. These results indicate that the cellular immunity to mycobacteria was recovering in the lung tissue. In M. parascrofulaceum pulmonary infection, the exudative lesion had characteristics of Th2 and Th17-type immunities. In contrast, the proliferative lesion had characteristics of Th-1 type immunity. Our data provide the first evidence to reveal the status of the axis of distinctive immunity in the process of granuloma formation caused by a mycobacterium-related infection.