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Buruli ulcer (BU) is a skin disease caused by Mycobacterium ulcerans infection that requires long-term antibiotic treatment and/or surgical excision. In this study, we investigated the therapeutic efficacy of the rifamycin derivative, rifalazil (RLZ) (also known as KRM-1648), in an advanced M. ulcerans infection model. Six-week-old female BALB/c mice were infected with 3.25 x 104 colony-forming units (CFU) of M. ulcerans subcutaneously into the bilateral hind footpads. At 33 days post-infection, when the footpads exhibited significant redness and swelling, mice were treated orally with 5 or 10 mg/kg of RLZ for up to 15 weeks. Mice were followed for an additional 15 weeks following treatment cessation. Untreated mice exhibited a progressive increase in footpad redness, swelling, and erosion over time, and all untreated mice reached to endpoint within 5-8 weeks post-bacterial injection. In the RLZ-treated mice, footpad redness and swelling and general condition improved or completely healed, and no recurrence occurred following treatment cessation. After 3 weeks of treatment, the CFU counts from the footpads of recovered RLZ-treated mice showed a 104 decrease compared with those of untreated mice. We observed a further reduction in CFU counts to the detection limit following 6 to 15 weeks of treatment, which did not increase 15 weeks after discontinuing the treatment. Histopathologically, bacteria in the treated mice became fragmented one week after RLZ-treatment. At the final point of the experiment, all the treated mice (5mg/kg/day; n = 6, 10mg/kg/day; n = 7) survived and had no signs of M. ulcerans infection. These results indicate that the rifamycin analogue, RLZ, is efficacious in the treatment of an advanced M. ulcerans infection mouse model., Competing Interests: No

It is important to select an appropriate surrogate matrix for preparing calibration standards and quality control samples while quantitatively assaying for endogenous substances, because a blank matrix that does not contain the endogenous substance cannot be derived from the species from which the target study samples are collected. This is because the assay results might be affected, depending on the characteristics of the analyte in the surrogate matrix. Our discussion group that participated in the Japan Bioanalysis Forum discussed the recommended selection strategies, focusing on large and small molecules in ligand binding assays and LC-MS, respectively. We established an efficient selection strategy for a surrogate matrix, with simple compositions as the first candidates stated in this article.

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.

Mycobacterium haemophilum is a slow-growing, non-tuberculous mycobacteria that causes cutaneous infection. We describe a case of cutaneous infection in a 68-year-old Japanese man with polymyositis. This was caused by M. haemophilum harboring one base insertion in gene sequence. At first, the causal microorganism was misidentified as M. intracellulare by COBAS ® TaqMan ® MAI test. However, poor growth on Ogawa media and growth enhancement on 7H11C agar around a hemin-containing disk prompted us to reinvestigate the causal microorganisms, which were revealed to be M. haemophilum. Amplified polymerase chain reaction products were sequenced, and the 16S rRNA gene, rpoB, hsp65 and internal transcribed spacer region sequences showed a 100%, 100%, 99.66% and 99.7% match, respectively, with the corresponding regions of M. haemophilum, but it harbored a novel gene sequence in hsp65. The sequences determined by gene analysis of the M. haemophilum strain were deposited into the International Nucleotide Sequence Database. Although numerous cases of M. haemophilum infection have been reported in other countries, only six cases have been reported in Japan to date. It could be possible that this novel mutation lead to misdiagnosis. As M. haemophilum prefers a lower growth temperature (30-32°C) and it requires iron in the culture medium, M. haemophilum could be misidentified or overlooked. Accordingly, a M. haemophilum infection should be considered in cases of cutaneous infection of the body sites, of which surface temperature is low., (© 2017 Japanese Dermatological Association.)

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., (Copyright © 2016 Yoshida et al.)

Purpose: We sought to gain a better understanding of the low-dose ionizing radiation (LDIR)-induced molecular changes in transformed pre-malignant cells in their microenvironment., Materials and Methods: The cellular response to LDIR was compared and contrasted using immortalized human Epstein-Barr virus-infected B-cells (EBV-B) in mono-culture, co-culture with human bone marrow derived stromal cells (MSC), or under the LDIR-induced bystander effect. The resulting alterations in protein and gene expression (including microRNA, miRNA) were evaluated by isobaric tags for relative and absolute quantification (iTRAQ) proteomics assay, western blot, cDNA array and quantitative reverse transcription polymerase chain reaction (RT-PCR), respectively., Results: The miRNAs let7a, miR-15b, miR-16, and miR-21, and a lipid metabolic miRNA hub miR-23b, were upregulated after LDIR exposure in the mono-cultured EBV-B cells, but were downregulated in EBV-B cells co-irradiated with MSC. A lipid biosynthesis enzyme glycerol-3-phosphate acyltransferase, the common target of these miRNA, was downregulated at the level of protein and mRNA expression in the LDIR-exposed, mono-cultured EBV-B cells and upregulated MSC co-cultured EBV-B cells., Conclusions: These results suggest a putative miRNA regulatory mechanism controlling the LDIR-induced stress response, and illustrate that LDIR exposure, and the cell's microenvironment, can affect specific gene expression, both directly and indirectly, resulting in altered protein expression.

Buruli ulcer (BU), or Mycobacterium ulcerans infection, is a new emerging infectious disease which has been reported in over 33 countries worldwide. It has been noted not only in tropical areas, such as West Africa where it is most endemic, but also in moderate non-tropical climate areas, including Australia and Japan. Clinical presentation starts with a papule, nodule, plaque or edematous form which eventually leads to extensive skin ulceration. It can affect all age groups, but especially children aged between 5 and 15 years in West Africa. Multiple-antibiotic treatment has proven effective, and with surgical intervention at times of severity, it is curable. However, if diagnosis and treatment is delayed, those affected may be left with life-long disabilities. The disease is not yet fully understood, including its route of transmission and pathogenesis. However, due to recent research, several important features of the disease are now being elucidated. Notably, there may be undiagnosed cases in other parts of the world where BU has not yet been reported. Japan exemplifies the finding that awareness among dermatologists plays a key role in BU case detection. So, what about in other countries where a case of BU has never been diagnosed and there is no awareness of the disease among the population or, more importantly, among health professionals? This article will revisit BU, reviewing clinical features as well as the most recent epidemiological and scientific findings of the disease, to raise awareness of BU among dermatologists worldwide., (© 2015 Japanese Dermatological Association.)

Mycobacterium haemophilum is a slow-growing non-tuberculous mycobacterium that is rarely known to cause human skin infection, particularly in immunocompromised patients. We recently experienced a 69-year-old Japanese woman with this infection who had been under immunosuppressive treatment for recalcitrant rheumatoid arthritis. The patient showed disseminated erythematous plaques and subcutaneous nodules on the face and extremities, and interestingly, the face manifested with a striking "facies leontina" appearance. Biopsy revealed abscess and granulomatous dermatitis with the involvement of peripheral nerve bundles and the presence of innumerable acid-fast bacilli, thus necessitating differentiation from lepromatous leprosy. M. haemophilum was identified by molecular characterization as well as by successful culture with iron supplements. Although drug susceptibility testing indicated responsiveness to multiple antibiotics administrated simultaneously for the treatment, it took over 6 months to achieve significant improvement, and we also employed concurrent oral potassium iodide administration and repeated surgical excision. This case highlights the importance of continuous combination therapy for successful outcome in this rare infection. Furthermore, application of potassium iodide for mycobacterial infection warrants further evaluation by accumulating more cases., (© 2015 Japanese Dermatological Association.)

Mycobacterium chelonae frequently involves the skin, and the disseminated form can be observed in immunocompromised patients. In contrast, rhinosinusitis caused by the bacterium is a rare manifestation, which occurs independently of immune status. We report here a rare case of M. chelonae infection presenting as both disseminated cutaneous infection and rhinosinusitis in an immunocompromised patient. He had received systemic corticosteroids for 11 months due to cryptogenic organizing pneumonia. Before admission, he sustained injuries to his left arm and hand; those injuries succumbed to an infection that would subsequently spread to his other limbs, face, and even nasal cavities. This valuable case suggests that disseminated cutaneous infection by M. chelonae could spread to other organs., (Copyright © 2015 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Buruli ulcer (BU) is a refractory skin ulcer caused by Mycobacterium ulcerans or M. ulcerans ssp. shinshuense, a subspecies thought to have originated in Japan or elsewhere in Asia. Although BU occurs most frequently in tropical and subtropical areas such as Africa and Australia, the occurrence in Japan has gradually increased in recent years. The World Health Organization recommends multidrug therapy consisting of a combination of oral rifampicin (RFP) and i.m. streptomycin (SM) for the treatment of BU. However, surgical interventions are often required when chemotherapy alone is ineffective. As a first step in developing a standardized regimen for BU treatment in Japan, we analyzed detailed records of treatments and prognoses in 40 of the 44 BU cases that have been diagnosed in Japan. We found that a combination of RFP (450 mg/day), levofloxacin (LVFX; 500 mg/day) and clarithromycin (CAM; at a dose of 800 mg/day instead of 400 mg/day) was superior to other chemotherapies performed in Japan. This simple treatment with oral medication increases the probability of patient adherence, and may often eliminate the need for surgery., (© 2015 Japanese Dermatological Association.)

Mitsugumin 56 (MG56), also known as the membrane-bound O-acyl-transferase family member hedgehog acyltransferase-like, was identified as a new sarcoplasmic reticulum component in striated muscle. Mg56-knockout mice grew normally for a week after birth, but shortly thereafter exhibited a suckling defect and died under starvation conditions. In the knockout skeletal muscle, regular contractile features were largely preserved, but sarcoplasmic reticulum elements swelled and further developed enormous vacuoles. In parallel, the unfolded protein response was severely activated in the knockout muscle, and presumably disrupted muscle development leading to the suckling failure. Therefore, MG56 seems essential for postnatal skeletal muscle maturation., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

Lysophospholipids (LysoGPs) serve as lipid mediators and precursors for synthesis of diacyl phospholipids (GPs). LysoGPs detected in cells have various acyl chains attached at either the sn-1 or sn-2 position of the glycerol backbone. In general, acyl chains at the sn-2 position of 2-acyl-1-LysoGPs readily move to the sn-1 position, generating 1-acyl-2-lyso isomers by a nonenzymatic reaction called intra-molecular acyl migration, which has hampered the detection of 2-acyl-1-LysoGPs in biological samples. In this study, we developed a simple and versatile method to separate and quantify 2-acyl-1- and 1-acyl-2-LysoGPs. The main point of the method was to extract LysoGPs at pH 4 and 4°C, conditions that were found to completely eliminate the intra-molecular acyl migration. Under the present conditions, the relative amounts of 2-acyl-1-LysoGPs and 1-acyl-2-LysoGPs did not change at least for 1 week. Further, in LysoGPs extracted from cells and tissues under the present conditions, most of the saturated fatty acids (16:0 and 18:0) were found in the sn-1 position of LysoGPs, while most of the PUFAs (18:2, 20:4, 22:6) were found in the sn-2 position. Thus the method can be used to elucidate the in vivo role of 2-acyl-1-LysoGPs., (Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.)

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are second-generation lysophospholipid mediators that exert multiple biological functions through their own cognate receptors. They are both present in the blood stream, activate receptors with similar structures (endothelial differentiation gene receptors), have similar roles in the vasculature and are vasoactive. However, it is unclear whether these lysophospholipid mediators cross-talk downstream of each receptor. Here, we provide in vivo evidence that LPA signaling counteracted S1P signaling. When autotaxin (Atx), an LPA-producing enzyme, was overexpressed in zebrafish embryos by injecting atx mRNA, the embryos showed cardia bifida, a phenotype induced by down-regulation of S1P signaling. A similar cardiac phenotype was not induced when catalytically inactive Atx was introduced. The cardiac phenotype was synergistically enhanced when antisense morpholino oligonucleotides (MO) against S1P receptor (s1pr2/mil) or S1P transporter (spns2) was introduced together with atx mRNA. The Atx-induced cardia bifida was prominently suppressed when embryos were treated with an lpar1 receptor antagonist, Ki16425, or with MO against lpar1. These results provide the first in vivo evidence of cross-talk between LPA and S1P signaling.

Importance: Buruli ulcer, a severe skin infectious disease caused by Mycobacterium ulcerans, is prevalent in tropical and subtropical areas. Recently, cases of Buruli ulcer have been increasing in Japan. All cases have been sporadic, and to date the pathogenic organism has not been detected in materials from the environment., Observations: Three members of the same family were seen with large indurated plaques on their face and extremities in the winter of 2010. Skin biopsy specimens of their lesions showed extensive necrosis of deep dermis and subcutaneous fat. Acid-fast bacilli were detected in each biopsy specimen by Ziehl-Neelsen stain, and bacteriological analysis of cultured microorganisms revealed the strains to be M ulcerans subsp shinshuense. The patients were treated with a combination of antibiotics and surgical debridement. Insertion sequence 2404 was detected from a crayfish captured in a stagnant water channel in the backyard of the family's house., Conclusions and Relevance: We report a rare instance of familial occurrence of Buruli ulcer in Japan. Detection of insertion sequence 2404 from a crayfish suggests that the pathogenic organism may reside in an aquatic environment in Japan, as in other endemic areas. To prevent this serious infectious disease, further investigation is needed to clarify the transmission pathway.

The rapidly growing mycobacterium M. abscessus sensu lato is the causative agent of emerging pulmonary and skin diseases and of infections following cosmetic surgery and postsurgical procedures. M. abscessus sensu lato can be divided into at least three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii. Clinical isolates of rapidly growing mycobacteria were previously identified as M. abscessus by DNA-DNA hybridization. More than 30% of these 117 clinical isolates were differentiated as M. abscessus subsp. massiliense using combinations of multilocus genotyping analyses. A much more cost-effective technique to distinguish M. abscessus subsp. massiliense from M. abscessus subsp. abscessus, a multiplex PCR assay, was developed using the whole-genome sequence of M. abscessus subsp. massiliense JCM15300 as a reference. Several primer sets were designed for single PCR to discriminate between the strains based on amplicons of different sizes. Two of these single-PCR target sites were chosen for development of the multiplex PCR assay. Multiplex PCR was successful in distinguishing clinical isolates of M. abscessus subsp. massiliense from samples previously identified as M. abscessus. This approach, which spans whole-genome sequencing and clinical diagnosis, will facilitate the acquisition of more-precise information about bacterial genomes, aid in the choice of more relevant therapies, and promote the advancement of novel discrimination and differential diagnostic assays.

There is evidence that the incidence of cutaneous non-tuberculous mycobacterial (NTM) infection is increasing worldwide. Novel culture methods and new analytical procedures have led to significant advancements in understanding the origin and progression of NTM infections. Differential identification of NTM isolates is important because culture characteristics and/or sensitivity to anti-mycobacterium drugs vary between different mycobacterial species. In this manuscript, we describe the latest diagnostic techniques for cutaneous NTM infection and show how these methodologies can be used for the diagnosis of Buruli ulcer in Japan., (© 2012 Japanese Dermatological Association.)

Buruli ulcer (BU) is an emerging human disease caused by Mycobacterium ulcerans, which mainly affects the extremities. It is most endemic in sub-Saharan Africa; however, it has been reported worldwide, including in some non-tropical areas. "M. ulcerans subsp. shinshuense" is proposed as a subspecies of M. ulcerans, which have been reported from Japan and China. A total of 35 BU cases have been reported as of November 2012. Although M. ulcerans is categorized as nontuberculous mycobacteria, it has some unique characteristics that could only be observed in this bacterium. It possesses a giant virulent plasmid, composed of 174-kbp nucleotides, coding polyketide synthase to produce macrolide toxin called mycolactone. The discovery of such a linkage of plasmid and its pathogenesis has not been reported in other human disease-causing mycobacteria.

PIWI-interacting RNAs (piRNAs) silence transposons to maintain genome integrity in animal germ lines. piRNAs are classified as primary and secondary piRNAs, depending on their biogenesis machinery. Primary piRNAs are processed from long non-coding RNA precursors transcribed from piRNA clusters in the genome through the primary processing pathway. Although the existence of a ribonuclease participating in this pathway has been predicted, its molecular identity remained unknown. Here we show that Zucchini (Zuc), a mitochondrial phospholipase D (PLD) superfamily member, is an endoribonuclease essential for primary piRNA biogenesis. We solved the crystal structure of Drosophila melanogaster Zuc (DmZuc) at 1.75 Å resolution. The structure revealed that DmZuc has a positively charged, narrow catalytic groove at the dimer interface, which could accommodate a single-stranded, but not a double-stranded, RNA. DmZuc and the mouse homologue MmZuc (also known as Pld6 and MitoPLD) showed endoribonuclease activity for single-stranded RNAs in vitro. The RNA cleavage products bear a 5'-monophosphate group, a hallmark of mature piRNAs. Mutational analyses revealed that the conserved active-site residues of DmZuc are critical for the ribonuclease activity in vitro, and for piRNA maturation and transposon silencing in vivo. We propose a model for piRNA biogenesis in animal germ lines, in which the Zuc endoribonuclease has a key role in primary piRNA maturation.

Bacteremia due to Mycobacterium massiliense is rare. We present here the case of a 58-year-old man with chronic myelogenous leukemia complicated by bacteremia caused by M. massiliense. The microorganisms were identified as M. massiliense by sequencing analysis, having initially been misdiagnosed as M. abscessus by DNA-DNA hybridization., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Buruli ulcer (BU) is a new emerging disease and the third most common chronic mycobacterial infection in humans, caused by Mycobacterium ulcerans. Approximately 5000 cases are reported annually from at least 33 countries around the globe, but more from the tropical nations. A total of 32 cases have been reported from Japan sporadically since 1980. None of the cases were related to international travel. Of the total reported, M. ulcerans ssp. shinshuense, a subspecies speculated to be domestic to Japan or in Asia, has been isolated from 23 cases. The mode of transmission and its incubation period remain unclear, despite several proposed hypotheses, including several vectors and cutaneous wound as port of entry for the pathogen. M. ulcerans invades the skin, subcutaneous tissue, fascia and eventually forms extensive ulceration. Smear, culture, histopathology and polymerase chain reaction are established diagnostic tools to identify M. ulcerans. Multiple antimicrobial therapy is a commonly used therapeutic method, but patients often need extensive debridement and, at times, skin grafting, especially when diagnosis is delayed. Thus, expanding a system for improved awareness and diagnosis in Japan and Asia is important, together with elucidating the candidate vector and the mode of transmission. Here, to establish a base for future progress in better understanding of this infectious disease, we reviewed the characteristics of the disease together with an update of reported cases in Japan., (© 2012 Japanese Dermatological Association.)

A novel slow-growing scotochromogenic mycobacterium was isolated from skin biopsies from a patient with a history of Hodgkin's disease and severe cellular immunodeficiency as an opportunistic pathogen. Clinical characterization of these lesions revealed papules and nodules with pathological granuloma formation. Genotypic analysis using 16S rRNA misidentified this isolate as Mycobacterium simiae. However, multiple gene analysis using the internal transcribed spacer between the 16S and 23S rRNA genes, and the rpoB and hsp65 genes revealed the presence of a novel mycobacterium. The antimicrobial susceptibility of this isolate was completely different from that of M. simiae. On the basis of these findings, we propose naming this new species Mycobacterium shigaense sp. nov., and conclude that multiple gene analysis is required for the appropriate diagnosis and treatment of non-tuberculous mycobacterial infections., (© 2011 Japanese Dermatological Association.)

Mycobacteria isolated from epizootics of farmed fishes in western Japan were examined for the first time using multigenotypic analysis. By analysis of the sequences of the internal transcribed spacer between the 16S and 23S rRNA genes (ITS) region and the partial 16S rRNA, hsp65 and rpoB genes, M. pseudoshottsii was identified as the causative agent in these infections. Prior to this study, only M. marinum has been known as the causative agent of lethal mycobacterial disease in marine fishes in Japan.

Autotaxin (ATX) is a multifunctional ecto-type phosphodiesterase that converts lysophospholipids, such as lysophosphatidylcholine, to lysophosphatidic acid (LPA) by its lysophospholipase D activity. LPA is a lipid mediator with diverse biological functions, most of which are mediated by G protein-coupled receptors specific to LPA (LPA1-6). Recent studies on ATX knock-out mice revealed that ATX has an essential role in embryonic blood vessel formation. However, the underlying molecular mechanisms remain to be solved. A data base search revealed that ATX and LPA receptors are conserved in wide range of vertebrates from fishes to mammals. Here we analyzed zebrafish ATX (zATX) and LPA receptors both biochemically and functionally. zATX, like mammalian ATX, showed lysophospholipase D activity to produce LPA. In addition, all zebrafish LPA receptors except for LPA5a and LPA5b were found to respond to LPA. Knockdown of zATX in zebrafish embryos by injecting morpholino antisense oligonucleotides (MOs) specific to zATX caused abnormal blood vessel formation, which has not been observed in other morphant embryos or mutants with vascular defects reported previously. In ATX morphant embryos, the segmental arteries sprouted normally from the dorsal aorta but stalled in midcourse, resulting in aberrant vascular connection around the horizontal myoseptum. Similar vascular defects were not observed in embryos in which each single LPA receptor was attenuated by using MOs. Interestingly, similar vascular defects were observed when both LPA1 and LPA4 functions were attenuated by using MOs and/or a selective LPA receptor antagonist, Ki16425. These results demonstrate that the ATX-LPA-LPAR axis is a critical regulator of embryonic vascular development that is conserved in vertebrates.

The etiology, clinical manifestations, and treatment of 19 sporadic cases of Buruli ulcer (BU) in Japan are described. The cases originated in different regions of Honshu Island, with no evidence of patient contact with an aquatic environment. The majority (73.7%) of cases occurred in females, with an average age of 39.1 years for females and 56.8 years for males. All patients developed ulcers on exposed areas of the skin (e.g., face, extremities). Most ulcers were <5 cm in diameter (category I), except in one severe progressive case (category II). Pain was absent in 10 of the 19 cases. Fourteen ulcers were surgically excised, and nine patients needed skin grafting. All cases were treated with various antibiotic regimens, with no reported recurrences as of March 2011. Mycobacterium ulcerans-specific IS2404 was detected in all cases. Ten isolates had identical 16S rRNA gene sequences, which were similar to those of M. ulcerans. However, the rpoB gene showed a closer resemblance to Mycobacterium marinum or Mycobacterium pseudoshottsii. PCR identified pMUM001 in all isolates but failed to detect one marker. DNA-DNA hybridization misidentified all isolates as M. marinum. The drug susceptibility profile of the isolates also differed from that of M. ulcerans. Sequence analysis revealed "Mycobacterium ulcerans subsp. shinshuense" as the etiologic agent of BU in Japan. Clinical manifestations were comparable to those of M. ulcerans but differed as follows: (i) cases were not concentrated in a particular area; (ii) there was no suspected connection to an aquatic environment; (iii) drug susceptibility was different; and (iv) bacteriological features were different.

Seven body polishers working in the same "hot spa" presented with multiple red nodules and papules on their hands and forearms. A causative agent was successfully isolated from two of the subjects and from a swab sample collected from the underside of a bed cover in the body-polishing facility. The two cutaneous isolates and the environmental isolate were rapidly growing mycobacteria that formed nonphotochromogenic smooth or smooth/rough colonies on Ogawa egg slants. They were identified as Mycobacterium massiliense by multigenotypic analysis using the 16S rRNA, hsp65, and rpoB genes and the 16S-23S rRNA internal transcribed spacer (ITS) region. However, the use of the 16S rRNA gene sequence and/or DNA-DNA hybridization (DDH Mycobacteria Kit) alone would not distinguish M. massiliense from mycobacteria in the M. chelonae-M. abscessus group. The three isolates were significantly more susceptible to clarithromycin, doxycycline, and minocycline than the M. abscessus and M. bolletii reference strains. One cutaneous isolate and the environmental isolate were in a related cluster by randomly amplified polymorphic DNA PCR (RAPD-PCR). Of the several mycobacterial species found in the day spa, only M. massiliense was isolated from biopsy specimens of the skin lesions, suggesting that this bacterium is a human skin pathogen. This is the first known report of cutaneous M. massiliense infections that could not be attributed to a prior invasive procedure. This is also the first report of M. massiliense infection in Japan.

A 75-year-old man with interstitial pneumonia due to ANCA-related vasculitis requiring immunosuppressive treatment was admitted to our hospital because of fever and rapidly progressive dyspnea. Chest CT showed diffuse ground-glass opacity with infiltration shadow in the bilateral lungs. We established a definitive diagnosis by isolating Mycobacterium massiliense on culture examination of acid-fast bacilli from peripheral blood and sputum. We began to administer CAM, LVFX, AMK, IPM/CS to this patient two weeks after admission. However, he died of respiratory failure and septic shock. There are few case reports of pulmonary lesion with septicemia due to Mycobacterium massiliense.

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.

Objective: To clarify the genetic microheterogeneity of Mycobacterium lentiflavum and identify the predominant genotype., Materials and Methods: Clinical isolates of M. lentiflavum used in this study were obtained from sixteen patients of lung diseases. In order to assess their intraspecies variability, four gene fragments, from the 16S rDNA (1471 bp), 16S-23S ITS (282 bp), rpoB (306 bp), and hsp65 (401 bp), were sequenced., Results: Intraspecies variabilities were found in all of the four targeting fragments. As multilocus sequence typing with these four targets, 16 clinical isolates were divided into 3 genotypes, i.e., MLST2, MLST3, and MLST4. Among them, MLST2 to which 12 clinical isolates belonged, was a predominant genotype. Three strains belonged to MLST3 and the remaining one strain belonged to MLST4. Drug susceptibility study indicated that there was no clear relation between sequence types and drug susceptibility., Conclusion: Multilocus sequence typing could aid in characterization and in better understanding of the epidemiology of M. lentiflavum.

Buruli ulcer is a chronic skin disease caused by Mycobacterium ulcerans, which produces a toxic lipid mycolactone. Despite the extensive necrosis and tissue damage, the lesions are painless. This absence of pain prevents patients from seeking early treatment and, as a result, many patients experience severe sequelae, including limb amputation. We have reported that mice inoculated with M. ulcerans show loss of pain sensation and nerve degeneration. However, the molecules responsible for the nerve damage have not been identified. In order to clarify whether mycolactone alone can induce nerve damage, mycolactone A/B was injected to footpads of BALB/c mice. A total of 100 microg of mycolactone induced footpad swelling, redness, and erosion. The von Frey sensory test showed hyperesthesia on day 7, recovery on day 21, and hypoesthesia on day 28. Histologically, the footpads showed epidermal erosion, moderate stromal edema, and moderate neutrophilic infiltration up to day 14, which gradually resolved. Nerve bundles showed intraneural hemorrhage, neutrophilic infiltration, and loss of Schwann cell nuclei on days 7 and 14. Ultrastructurally, vacuolar change of myelin started on day 14 and gradually subsided by day 42, but the density of myelinated fibers remained low. This study demonstrated that initial hyperesthesia is followed by sensory recovery and final hypoesthesia. Our present study suggests that mycolactone directly damages nerves and is responsible for the absence of pain characteristic of Buruli ulcer. Furthermore, mice injected with 200 microg of mycolactone showed pulmonary hemorrhage. This is the first study to demonstrate the systemic effects of mycolactone.

We describe the fourth reported case involving "Mycobacterium ulcerans subsp. shinshuense." Compared to previous cases, the infection was more invasive with extensive ulceration from the elbow to the forearm. Definitive identification involved IS2404 detection, 16S rRNA gene sequencing, and analysis of the 16S rRNA gene 3'-terminal region and the virulence plasmid pMUM001.

Although Mycobacterium shinshuense and M. leprae infections are relatively rare in the fields of dermatology, an early diagnosis is one of the important prognostic factors of these infections. Applications of the genetical examinations such as PCR and 16S rDNA sequencing are helpful in early diagnosis with culture nagative cases. Short target PCR tests are available to detect DNA of M. shinshuense or M. leprae from clinical specimens including formalin fixed-paraffin embedded samples. A partial 16s rDNA sequencing is functional with enough intact bacterial DNA. A similarity search based on the partial 16S rDNA sequences using RIDOM database is an easy and powerful tool to estimate the species of mycobacteria, however, it is not enough for the identification in some cases. For instance, a clinical isolate of M. shinshuense is clearly discriminated from M. leprae (92.75% sequence identity), however, difficult to be identified from M. marinum and M. ulcerans (99.77% sequence identity). The phylogenetic tree based on 16S rDNA sequences is illustrating that both M. leprae (closely related to M. haemophilum) and M. shinshuense (closely related to M. marinum and M. ulcerans, and also M. tuberculosis) are relatively related species and distantly related to rapidly growing species among 30 species of pathogenic mycobacteria which have been isolated in Japan.

Mycobacterium shimoidei (Tsukamura 1982) is an uncommon but widely distributed pathogen usually isolated from respiratory specimens. We report two cases of lung disease due to M. shimoidei and the associated bacteriological results. A 45-year-old man (Case 1) admitted to National Hospital Organization (NHO) Miyagi Hospital, a 75-year-old man (Case 2) admitted to NHO Higashi-Hiroshima Medical Center were found in initial chest X-ray and thoracic computed tomography (CT) to have a tuberculosis-like cavity in the left apex (Case 1) and the right apex (Case 2). In Case 1, the patient was treated with isoniazid and rifampicin for one month and lesions showed a partial response. In Case 2, the patient responded favorably with rifampicin, ethambutol, streptomycin, and clarithromycin therapy. Mycobacteria were repeatedly detected in smear and culture from sputum specimens in both patients. Isolates were nonphotochromogenic and rough. Isolated colonies developed after two to three weeks on 2% Ogawa egg medium. Organisms grew on 2% Ogawa egg medium at 30, 37, 42, and 45 degrees C, but not 25 degrees C. Both organisms were susceptible to 500 microg of p-nitrobenzoate per mL and 5mg of sodium chloride per mL. Isolates were negative for niacin accumulation, nitrate reduction, semiquantitative catalase, 68 degrees C catalase, 3-day aryl-sulfatase, iron uptake, and MPB64 antigen production, but positive for Tween 80 hydrolysis (5 and 10 days), acid phosphatase, and pyrazinamidase. Isolates had typical uv-HPLC chromatograms similar to M. shimoidei, demonstrating triple-peak clusters with peaks in the early cluster. 16S rRNA gene sequencing showed isolates to be consistent with Mycobacterium shimoidei. Based on composite characterization, isolates were identified as M. shimoidei. This is, to our knowledge, the third case of M. shimoidei infection reported in Japan.

Laboratory tests necessary for the diagnosis of leprosy have not been well introduced in general hospitals and clinical laboratories. Therefore, several tests have been performed in Leprosy Research Center, National Institute of Infectious Diseases since July, 1997, as a part of administrative examinations (tests done by request of Ministry of Health, Labour and Welfare). These examinations include histopathology, serum antibody titers (anti-PGL-I antibody), PCR test and bioactivity of anti-bacterial agents.

Buruli ulcer is an emerging chronic painless skin disease found in the tropics and caused by Mycobacterium ulcerans; however, it remains unknown why the large and deep ulcers associated with this disease remain painless. To answer this question, we examined the pathology of BALB/c mice inoculated in the footpads with M. ulcerans African strain 97-107. On days 54 to 70 after inoculation, extensive dermal ulcers, subcutaneous edema, and numerous acid-fast bacilli were noted at the inoculate region. Nerve invasion occurred in the perineurium and extended to the endoneurium, and some nerve bundles were swollen and massively invaded by acid-fast bacilli. However, Schwann cell invasion, a characteristic of leprosy, was not observed. Vacuolar degeneration of myelin-forming Schwann cells was noted in some nerves which may be induced by mycolactone, a toxic lipid produced by M. ulcerans. Polymerase chain reaction analysis of microdissected nerve tissue sections showed positive amplification of M. ulcerans-specific genomic sequences but not of Mycobacterium leprae-specific sequences. Behavioral tests showed decrease of pain until edematous stage, but markedly ulcerated animals showed ordinary response against stimulation. Our study suggests that the painlessness of the disease may be partly due to intraneural invasion of bacilli. Further studies of nerve invasion in clinical samples are urgently needed.