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).

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).

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.

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.

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.

[Non-tuberculous mycobacterium strains that show positive test for identification kits of M. tuberculosis complex].

OBJECTIVES: Saito et al. isolated novel mycobacterium strains from the sputum of 12 patients with pulmonary disease. They reported, that the strains were clearly different from Mycobacterium tuberculosis (TB) in cultural, biochemical and immunological properties, despite the high homology (99.1%) of the 16S rRNA gene sequence between the two. Recently, we isolated four strains having similar properties as the above strains among mycobacterial strains that were sent to the Research Institute of Tuberculosis for identification. We examined these isolates using commercial systems for identification of mycobacteria. MATERIALS: Four strains of the unidentified mycobacteria were used in this study. METHODS: Tests used in the study included cultural on solid media, biochemical characteristics, DNA sequence analyses of 16S rRNA and rpoB genes, TRC, COBAS AMPLICOR, COBAS TaqMan, MTD, DDH, Accu-Probe, Capilia TB, and a drug susceptibility tests. RESULTS: After three weeks of culture, smooth and non-photochromogenic colonies were formed. The niacin accumulation test was negative. The homologies of DNA sequence between the new strains and M. tuberculosis for 16S rRNA and rpoB genes were 97.8% and 90.2%, respectively. The tests with TRC and MTD kits were positive, whereas the tests with AMPLICOR, TaqMan, Accu-Probe and Capilia TB kits were negative. The organism was not identified with the DDH system.

[Clinical analysis of extensively-drug resistant tuberculosis (XDR-TB) in our hospital].

OBJECTIVE: We analyzed the clinical characteristics of extensively-drug resistant tuberculosis (XDR-TB). MATERIALS AND METHODS: Thirteen cases diagnosed with XDR-TB encountered in our hospital over the last ten years were enrolled in our study. RESULTS: The patients included 9 males and 4 females. The mean age was 49.1 years old in males and 42.0 years old in females. Eight patients were Japanese and 5 were foreigners (Chinese, 3; Korean, 1; Nepali, 1). Nine cases had a smoking history and 6 had underlying diseases, including 1 with bacterial pneumonia, 3 with diabetes mellitus, 1 with chronic renal failure, and 1 with collagen vascular disease receiving immunosuppressive treatment. All 13 cases had been diagnosed at other hospitals. The mean period from TB diagnosis to XDR-TB diagnosis was 56.8 months, and the mean period from TB diagnosis to referral to our hospital was 81.6 months. Among the 13 cases, 3 had no drug sensitivity, 1 was sensitive to only 1 drug, 2 were sensitive to 2 drugs, 6 were sensitive to 3 drugs, and 1 was sensitive to 4 drugs. Nine of the 13 cases had surgical treatment. Six cases, all of whom had surgical treatment, showed negative conversion in sputum examinations. Three patients died, including two who had surgical treatment. Among the 3 cases with no drug sensitivity, 1 was cured after surgical treatment. Another case had been working in the same hospital with two other MDR-TB cases. Two of the three had the same RFLP pattern. CONCLUSION: XDR-TB and MDR-TB are man-made diseases. We need to take measures not to create more XDR strains and induce more MDR-TB cases.

[Usefulness of the variable numbers of tandem repeats (VNTR) analysis for complex infections of Mycobacterium avium and Mycobacterium intracellulare].

PURPOSE: The bacilli which were isolated from a patient suspected of the mixed infections with Mycobacterium avium and Mycobacterium intracellulare, were analyzed. The genotypes of M. avium in the sedimented fractions of treated sputum and in some colonies isolated from Ogawa medium were compared by the Variable Numbers of Tandem Repeats (VNTR). CASE: A woman, aged 57. Mycobacterial species isolated from some colonies by culture in 2004 and 2006 and from the treated sputum in 2006, were determined by DNA sequencing analysis of the 16S rRNA gene. Also, by using VNTR, the genotype of mycobacteria was analyzed. [Results] (1) The colony isolated from Ogawa medium in 2004 was monoclonal M. avium. (2) By VNTR analyses of specimens in 2006, multiple acid-fast bacteria were found in the sputum sediment and in isolated bacteria from Ogawa medium. (3) By analyses of 16S rRNA DNA sequence, M. avium and M. intracellulare were found in the colonies isolated from the sputum sediment and the Ogawa medium in 2006. (4) The same VNTR patterns were obtained in M. avium in 2004 and 2006 when single colony was analyzed. (5) From the showerhead and culvert of the bathroom in the patient's house, M. avium was not detected. DISCUSSION: By VNTR analyses, it was considered that the mixed infections of M. avium and M. intracellulare had been generated during treatment in this case. Therefore, in the case of suspected complex infection, VNTR analysis would be a useful genotyping method in M. avium complex infection.

[Microbiological properties of M. porcinum isolated from a patient with impairments in IL-12/IFN-gamma pathway].

PURPOSE: Mycobacterium porcinum has been successfully isolated from the patient with abnormal signal transduction pathway of IL12/IFN-gamma. The properties of each bacterium were determined by conventional identification methods, DNA sequencing analysis and MIC assay. MATERIALS AND METHODS: M. porcinum was isolated 7 times from 1996 to 2007 from cervical lymph node, axillary lymph nodes, inguinal lymph node, brachial lymph node and site of a tumor of the patient. In another occasion, mycobacteria were isolated from lavage fluid of the endoscope in routine inspection. Using these mycobacteria, M. porcinum (ATCC33776) and M. fortuitum (ATCC6841), the conventional identification method and MIC assay were carried out. For analyses of the DNA sequencing (rpoB, dnaJ and hsp65), the ATCC type strain of mycobacteria (11 strains) which are closely related to M. porcinum were also used. RESULTS AND DISCUSSION: DNA sequencing analyses of the 7 samples isolated from the patient, were concurrently identical in 3 different genes. Drug susceptibility test showed that 7 isolates had no marked change. In conventional identification analyses, M. porcinum (ATCC33776), M. fortuitum (ATCC6841), and M. porcinum that were isolated in 1996, were able to grow at 42 degrees C. However, 6 isolates that were isolated after 1999, did not grow at 42 degrees C. The colony detectable days of these 7 strains changed from 3 to 7. Over the time, the morphology of each colony changed from smooth to rough. Though the initial isolate had the ability to utilize mannitol, the later 4 isolates had no such ability.

[Identification of 23 mycobacterial species by Invader assay with targeting 16S rRNA gene and ITS-1 region--comparison with DDH method in clinical isolates].

PURPOSE AND METHOD: The Invader assay was developed to identify 23 mycobacterial species using probes derived from the species-specific region of the 16S rRNA gene and the 16S-23S rRNA internal transcribed spacer 1 (ITS-1) region, with minor modifications of our previous study. In the present study, we compared the identification capability between the Invader assay and DNA-DNA hybridization (DDH) method. DDH is commonly used to identify non-tuberculosis mycobacterium in Japan and 636 clinical mycobacterial strains cultured on Ogawa slants were tested. RESULTS: The Invader assay could identify 615 (96.7%) of the 636 strains. The results contained 14 M.lentiflavum, 3 M. parascrofulaceum and 1 M. intermedium, which were undetectable with DDH method. On the other hand, DDH method could identify 580 (91.2%) strains with duplicate assay. Of 628 strains except 8 strains identified as a few species by Invader assay, 551 (87.7%) strains were identified as the same species by two methods. Discordant results were mainly recognized for the identification of M. gordonae, M. avium, M. lentiflavum and M. intracellurare. The results of other methods targeting 16S rRNA indicated correctness of the Invader assay. CONCLUSION: These results indicate that Invader assay could identify more correctly than DDH method and could identify about 97% of clinically important mycobacterium.

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.

Detection of streptomycin resistance in Mycobacterium tuberculosis clinical isolates from China as determined by denaturing HPLC analysis and DNA sequencing.

China is regarded by the World Health Organization as a major hot-spot region for Mycobacterium tuberculosis infection. Streptomycin has been deployed in China for over 50 years and is still widely used for tuberculosis treatment. We have developed a denaturing HPLC (DHPLC) method for detecting various gene mutations conferring drug resistance in M. tuberculosis. The present study focused on rpsL and rrs mutation analysis. Two hundred and fifteen M. tuberculosis clinical isolates (115 proved to be streptomycin-resistant and 100 susceptible by a routine proportional method) from China were tested to determine the streptomycin minimal inhibitory concentration (MIC), and subjected to DHPLC and concurrent DNA sequencing to determine rpsL and rrs mutations. The results showed that 85.2% (98/115) of streptomycin-resistant isolates harbored rpsL or rrs mutation, while rpsL mutation (76.5%, 88/115) dominated. MIC of 98 mutated isolates revealed no close correlation between mutation types and levels of streptomycin resistance. No mutation was found in any of the susceptible isolates. The DHPLC results were completely consistent with those of sequencing. The DHPLC method devised in this study can be regarded as a useful and powerful tool for detection of streptomycin resistance. This is the first report to describe DHPLC analysis of mutations in the rpsL and rrs genes of M. tuberculosis in a large number of clinical isolates.

[Case of pulmonary Mycobacterium mageritense infection: the difficulty of differential diagnosis of granulomatous lung diseases].

A 36 year-old female was pointed out of pulmonary abnormal shadows in the annual chest survey. Chest radiograph and computed tomography (CT) disclosed bilateral diffuse infiltrative shadows and tree-in-bud appearance in the right upper lung field and the left lingula. A sputum smear for acid-fast bacilli was negative. Histopathologically, the transbronchial lung biopsy specimen revealed non-caseous epithelioid granulomas with numerous giant cells. Acid-fast bacilli were cultured from her sputum, however, nontuberculous mycobacteria was not detected by DNA-DNA hybridization method. Mycobacterium mageritense was identified by 16S ribosomal RNA sequencing with 100% matching. The isolated colony of M. mageritense was resistant to nine anti-tuberculous drugs. Follow-up chest CT scan showed a gradual decrease of infiltrative shadows without therapy. To the best of our knowledge, M. mageritense infections are rare, and this is the first case report of pulmonary infection in the literature. We conclude that the pulmonary infection of M. mageritense is one of causes of granuloma formation, and in some case it is difficult to differentiate clinically from sarcoidosis.

[Comparison of usefulness between variable numbers of tandem repeats (VNTR) analysis and restriction fragment length polymorphism (RFLP) in the genotyping of Mycobacterium avium].

OBJECTIVES: Comparison of usefulness of IS1245 RFLP and VNTR in M. avium genotyping. MATERIALS AND METHODS: Thirty-six cases (55 strains) from sputum and BALF and twelve cases (29 strains) isolated from blood of HIV-infected patients were used. VNTR and RFLP using IS1245 were performed. RESULT: Multiple samples were taken from 16 patients and 52 clinical isolates were used for VNTR and RFLP for comparison. (1) VNTR and RFLP results were identical in 12 out of 16 cases whose samples were collected several times. (2) Eight isolates were obtained from one patient. In this eight isolates, there were the cases of M. avium polyclonal infection and of mixed infection with M. intracellulare. VNTR patterns were two types and RFLP were 5 kinds of different in this case. (3) VNTR patterns of six isolates from one HIV-infected patient were identical, but there were three variations in RFLP patterns. There were three cases of mixed infections with M. tuberculosis or M. intracellulare, and six strains polyclonal infection of M. avium (7.1 %) in 84 isolates. These 6 clinical isolates were derived from sputum or BALF (5 strains) and HIV-infected blood (one strain). VNTR patterns were similar in four pairs (9 strains) who did not contact closely, but they were distinguished clearly by RFLP. Seventeen strains had three or less IS1245-related bands in RFLP analyses of 89 strains. DISCUSSION: As there is a possibility of polyclonal infection with M. avium and mixed infection with other species, the single clonal infection should be confirmed first by VNTR. When single colony was obtained, VNTR and RFLP were performed for genotyping of M. avium. Furthermore, strains with less bands by RFLP should be carefully judged in terms of both VNTR and RFLP. It is recommended that the specimens should be collected from each patient several times.

[Frequency of MDR-TB/XDR-TB strains isolated from chronic pulmonary tuberculosis patients in Japan].

PURPOSE: To observe the frequency of MDR-TB/XDR-TB strains isolated from chronic pulmonary tuberculosis patients in Japan. OBJECT: Ad hoc National Tuberculosis Survey 2000 on frequency of MDR-TB and XDR-TB strains. MATERIALS AND METHOD: Four hundred and thirty four clinical isolates were collected by the Ad hoc National Tuberculosis Survey 2000, the drug susceptibility testings (proportion method, MGIT Middlebrook, and BrothMIC NTM) were conducted on these strains. These clinical isolates were obtained from patients registered at Health Centers in Japan by the end of 1999 who were culture-positive in 1999 and were registered before January 1st, 1998. The isolates used in this study were selected from patients who were culture-positive at shortest 2 years after the registration. RESULT: The clinical isolates resistant to both INH and RFP were 321 out of 434 (74.0%). The 180 MDR-resistant clinical isolates were also resistant to levofloxacin and amikacin and/or kanamycin. These phenotypes are XDR-TB. No previously registered cases were 165, and previously registered cases were 143 and unknown cases were 13 out of 321 MDR-TB. In 180 XDR-TB cases, no previously registered cases were 95, previously registered cases were 78 and unknown cases were 7. In no previously registered cases, more than 50% cases started treatment in 1990s. Approximately 50% of previously registered patients started treatment in 1960s and 1970s. CONCLUSION: We performed drug susceptibility testing for 434 clinical isolates which were culture-positive at shortest 2 years after registration. No. of MDR-TB patients was 321 and that of XDR-TB patients was 180. The treatment outcome of these patients have to be followed up carefully at Health Centers. The frequency of amikacin resistance was relatively high. This may be due to either common use of amikacin or cross-resistance against streptomycin and kanamycin.