[Show abstract][Hide abstract] ABSTRACT: 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 Unite 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
PLoS ONE 12/2014; 9(12):e114848. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We evaluated the anti-tuberculosis (TB) activity of five β-lactams alone or in combination with β-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 0.125-8 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.
Antimicrobial Agents and Chemotherapy 09/2014; · 4.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: Mycobacterium florentinum is a newly identified, rare, slow-growing species of nontuberculous mycobacteria (NTM). Here, we report a case of M. florentinum-induced synovitis of the wrist in an immunocompromised Japanese patient. M. florentinum was identified by sequence analysis of the rpoB, hsp65, and 16S rRNA genes. The M. florentinum strain in this study could not be differentiated from certain M. triplex strains by the hsp65 or 16S rRNA sequences alone, because they occasionally shared more than 99 % sequence identity. The isolated M. florentinum strain was only susceptible to clarithromycin and amikacin. Initially, the patient was treated with clarithromycin, levofloxacin, and ethambutol, and then with clarithromycin, levofloxacin, and rifampicin. To our knowledge, M. florentinum-induced synovitis has not been previously reported. Our results suggest that, in addition to other well-known pathogenic NTM, the recently identified M. florentinum strain should be considered as a possible cause of synovitis. Moreover, we should be cautious when identifying M. florentinum because this strain closely resembles M. triplex in genotype.
[Show abstract][Hide abstract] ABSTRACT: 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.
Kansenshogaku zasshi. The Journal of the Japanese Association for Infectious Diseases 07/2013; 87(4):424-30.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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 β-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.MethodsA 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).ResultsIn 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.
International Journal of Mycobacteriology. 03/2012; 1(1):21–28.
[Show abstract][Hide abstract] ABSTRACT: 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.
Internal Medicine 01/2012; 51(16):2203-7. · 0.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Pathology - Research and Practice 03/2011; 207(4):262-70. · 1.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Internal Medicine 01/2011; 50(11):1251-3. · 0.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We analyzed the clinical characteristics of extensively-drug resistant tuberculosis (XDR-TB).
Thirteen cases diagnosed with XDR-TB encountered in our hospital over the last ten years were enrolled in our study.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: Recent genetic studies have revealed that several epidemiological factors affect Mycobacterium avium complex (MAC) infection in pig populations. However, mechanisms underlying the spread of MAC infection among hog farms have not been clarified. In consideration of this situation, we cross-sectionally investigated the mechanisms underlying the spread of MAC on the island of Okinawa. Pigs slaughtered (n = 706,763) and 331 hog farms on Okinawa were surveyed during the years 2002–2004. Two outbreaks of MAC infection were occurred in several farms during survey period. Bacteria were isolated from randomly selected pigs and genotype of isolates was determined by using genetic finger printing methods with the insertion sequence (IS) 1245 restriction fragment length polymorphism (RFLP). Most isolates had large numbers of IS1245 copies, while strains with low copy numbers of IS1245 and isolates without IS1245 were seen in few farms. MACs strains were repeatedly isolated from pigs of the affected farms during the survey period. Those farms with an identical pig rearing systems showed synchronic changes in the prevalence of MAC infection. An industrial farm without an outbreak had an independent pig flow, but maintained distinct MAC strains. Multivariate analysis did not reveal independent factors for the prevalence of the MAC infection. These findings suggest that there were three clusters distinguished genetically in the main island of Okinawa, which were potentially spread by common pig flow. However, the outbreaks occurred because of unspecified conditions on each farm environment.RésuméDe récentes études en génétique ont dévoilé que plusieurs facteurs épidémiologiques ont des répercussions néfastes sur le complexe Mycobacterium avium (MAC) des porcs, ce qui les rend malades. Cependant, les mécanismes sous-jacents à la diffusion de l’infection du MAC dans les élevages de porcs n’ont pas été clairement identifiés. Par conséquent, nous avons examiné les mécanismes latents à la propagation du MAC sur l’île d’Okinawa en découpant la population totale des porcs en groupes d’animaux présentant les mêmes caractéristiques. Notre étude recense donc 706 763 bêtes réparties dans 331 fermes. Sur toute la période de nos travaux, le taux moyen des infections du MAC atteind au maximum 9,2% des animaux. Les fermes présentant des systèmes d’élevages identiques ont montré des modifications simultanées dans la fréquence des infections, et les organismes aux mêmes caractéristiques génétiques ont été séparés des porcs de ces fermes.Une ferme d’élevage en batterie dont les animaux proviennent d’un marché indépendant ne présente aucun pic d’infection, et garde des distinctions génétiques au niveau du MAC. Ces conclusions suggèrent qu’il y avait trois groupes distingués génétiquement dans l’île principale d’Okinawa qui s’est été étendu potentiellement par courant du cochon commun. Cependant, les premières manifestations se sont produites à cause de conditions non spécifiées sur chaque environnement de ferme.
[Show abstract][Hide abstract] ABSTRACT: 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.
Four strains of the unidentified mycobacteria were used in this study.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: To better understand nontuberculous mycobacteria (NTM) contamination in a hospital setting, six freshwater fish gut homogenates and water in an aquarium fish tank placed on the reception counter of a nursing station were cultured for mycobacteria.
By direct sequencing of 16s rRNA, rpoB and hsp65, scotochromogenic and nonchromogenic Mycobacterium szulgai isolates containing hsp65 type II (GenBank accession nos. FJ384762 and FJ384764, respectively), Mycobacterium gordonae isolates containing rpoB clusters B and E (GenBank accession no. FJ384766), and Mycobacterium kansasii isolates containing hsp65 type VI were collected from the gut homogenates and water from the fish tank. However, no isolates were obtained from the tap water used to refill the fish tank. A randomly amplified polymorphic DNA (RAPD) analysis using a 10-mer primer (5'-TGGTCGCGGC) showed that some NTM from the fish tank water were identical to those obtained from the gut homogenates.
Fish and water in the tank were contaminated by the novel NTM.
These findings could help to elucidate infection routes and contamination sources of novel NTM from water sources.
Journal of Applied Microbiology 02/2010; 109(2):558-66. · 2.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nontuberculous mycobacterial (NTM) infection in HIV (human immunodeficiency virus)-infected patients who have started highly active antiretroviral therapy (HAART) is well known to be one scenario of immune reconstitution inflammatory syndrome (IRIS). We encountered the first case in Japan of an HIV-infected patient with pulmonary Mycobacterium parascrofulaceum infection as IRIS. A 34 year-old man with acquired immunodeficiency syndrome (AIDS) was receiving highly active antiretroviral therapy. Lymphadenopathy was observed at the left pulmonary hilum. IRIS was suspected and thoracoscopic surgery was performed to diagnose the cause of lymphadenopathy. Granulomas were observed histologically, and M. parascrofulaceum was cultured. This organism was susceptible to Clarithromycin, rifampicin and levofloxacin. After the operation and without treatment, recurrence of M. parascrofulaceum infection was not observed. M. parascrofulaceum was isolated from several clinical specimens for the first time in 2004. To date, only five cases have been reported.
Internal Medicine 01/2010; 49(16):1817-21. · 0.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An extremely rare case of intractable ulcer caused by Mycobacterium shinshuense is described. A 59-year-old Japanese woman developed an ulcerated subcutaneous induration on the upper arm. Ziehl-Neelsen staining revealed positive bacilli. Tissue culture isolated Mycobacterium species, but standard identification techniques (including molecular biological approaches such as DNA-DNA hybridization) could not distinguish the precise causative pathogen, although it was narrowed down to three possibilities: Mycobacterium marinum, Mycobacterium ulcerans and M. shinshuense. Finally, a novel 16S rRNA sequencing method enabled the diagnosis of M. shinshuense infection. The epidemiology of the cutaneous infection caused by this mycobacterium has yet to be elucidated, but a review of reported cases indicated that ulcers having some resemblance to those caused by M. ulcerans infection were found in nonendemic areas and that M. shinshuense could be considered as the cause. The approach introduced in this report could provide a powerful tool for the identification of this organism.
Clinical and Experimental Dermatology 08/2009; 34(8):e712-5. · 1.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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).
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
These results indicate that Invader assay could identify more correctly than DDH method and could identify about 97% of clinically important mycobacterium.