Yuko Kazumi

Emergence of Ofloxacin Resistance in Mycobacterium tuberculosis Clinical Isolates from China as Determined by gyrA Mutation Analysis Using Denaturing High-Pressure Liquid Chromatography and DNA Sequencing

ABSTRACT A high rate of double point mutations in gyrA (56% of 87 ofloxacin-resistant Mycobacterium tuberculosis clinical isolates) indicates the emergence of fluoroquinolone resistance. This is the first report to describe denaturing high-pressure liquid chromatography analysis of mutations in gyrA of M. tuberculosis in a large number of clinical isolates.

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

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

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

ABSTRACT We evaluated the antituberculosis (anti-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 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.

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

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

Complete Genome Sequence of Mycobacterium ulcerans subsp. shinshuense

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

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

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

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

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

Pulmonary Mycobacterium intermedium Disease in an Elderly Man with Healed Pulmonary Tuberculosis

ABSTRACT A 76-year-old man with a history of pulmonary tuberculosis was found to be sputum smear positive for acid-fast bacilli. The 16S rRNA sequence identified the culture isolate as Mycobacterium intermedium, the pathogenicity of which has not been confirmed. Chemotherapy with isoniazid, rifampin, and ethambutol resulted in clinical improvement.

非結核性抗酸菌 （Mycobacterium chelonae chemovar niacinogenes） による院内感染事例の発生と拡大原因の考察

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.

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.