Therapeutic efficacy of rifalazil (KRM-1648) in a M. ulcerans-induced Buruli ulcer mouse model.

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.

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.

Case of Mycobacterium haemophilum misdiagnosed as Mycobacterium intracellulare due to one base insertion in the bacterial genome.

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.

Complete Genome Sequence of Mycobacterium ulcerans subsp. shinshuense.

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

Revisiting Buruli ulcer.

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.

Rhinosinusitis and disseminated cutaneous infection caused by Mycobacterium chelonae in an immunocompromised patient.

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.

Mycobacterium haemophilum infection with prominent facial manifestation mimicking leprosy.

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.

Exploration of a standard treatment for Buruli ulcer through a comprehensive analysis of all cases diagnosed in Japan.

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.

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

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

Buruli ulcer caused by Mycobacterium ulcerans subsp shinshuense: a rare case of familial concurrent occurrence and detection of insertion sequence 2404 in Japan.

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.

Discrimination of Mycobacterium abscessus subsp. massiliense from Mycobacterium abscessus subsp. abscessus in clinical isolates by multiplex PCR.

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.

Buruli ulcer and mycolactone-producing mycobacteria.

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.

Laboratory procedures for the detection and identification of cutaneous non-tuberculous mycobacterial infections.

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.

Bacteremia due to Mycobacterium massiliense in a patient with chronic myelogenous leukemia: case report.

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.

A Case of Mycobacterial Skin Disease Caused by Mycobacterium peregrinum, and a Review of Cutaneous Infection.

An 83-year-old Japanese man presented with a 2-month history of symptomatic nodules on the left hand. He was not in an immunocompromised condition and reported no causal events. A biopsy specimen demonstrated granulomatous tissue with mixed cell infiltration consisting of neutrophils, histiocytes, lymphocytes, and multinuclear giant cells. No bacillus was detected by PAS, acid-fast stain, immunofluorescent stain or polymerase chain reaction analysis. The isolate was found to be a rapidly growing mycobacterium after 4 weeks of incubation at 25°C on an Ogawa egg slant. Mycobacterium peregrinum was isolated by DNA-DNA hybridization analysis, 16S rRNA gene sequence, and by its production of 3-day arylsulfatase. The patient received 200 mg oral minocycline for 28 weeks. The lesion disappeared after 10 weeks of this treatment.

Buruli ulcer and current situation in Japan: a new emerging cutaneous Mycobacterium infection.

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.