EM, EM703 inhibit NF-kB activation induced by oxidative stress from diesel exhaust particle in human bronchial epithelial cells: importance in IL-8 transcription.

Diesel exhaust particle (DEP) is the major components of PM2.5, and much attention has focused on PM2.5 in relation to adverse health effects, and many pulmonary diseases. In the present study, we used a human bronchial epithelial cell (HBEC) line to investigate the anti-inflammatory effects of erythromycin (EM) and EM703 - a new derivative of erythromycin without antibacterial effects on the expressions of IL-8 caused by DEP exposure. DEP showed a dose-dependent stimulatory effect on IL-8 product in HBEC. Increases of IL-8 expression by DEP stimulation were significantly blocked by both EM and EM703 pretreatment. Furthermore, NF-κB and Nrf2 activation, the antioxidant enzymes such as HO-1, NQO-1 mRNA expression were increased by DEP exposure and these increases were blocked by both of EM and EM703 pretreatment. Our results suggest that, EM and EM703 may have an inhibitory effect on expression inflammatory cytokines in HBEC induced by DEP not only as an anti-inflammation but also an antioxidant drug. EM and EM703 might contribute to chemical prevention of the risk of pulmonary diseases induced by oxidative stress from environmental pollutant, such as DEP.

Mycobacteria exploit host hyaluronan for efficient extracellular replication.

In spite of the importance of hyaluronan in host protection against infectious organisms in the alveolar spaces, its role in mycobacterial infection is unknown. In a previous study, we found that mycobacteria interact with hyaluronan on lung epithelial cells. Here, we have analyzed the role of hyaluronan after mycobacterial infection was established and found that pathogenic mycobacteria can grow by utilizing hyaluronan as a carbon source. Both mouse and human possess 3 kinds of hyaluronan synthases (HAS), designated HAS1, HAS2, and HAS3. Utilizing individual HAS-transfected cells, we show that HAS1 and HAS3 but not HAS2 support growth of mycobacteria. We found that the major hyaluronan synthase expressed in the lung is HAS1, and that its expression was increased after infection with Mycobacterium tuberculosis. Histochemical analysis demonstrated that hyaluronan profoundly accumulated in the granulomatous legion of the lungs in M. tuberculosis-infected mice and rhesus monkeys that died from tuberculosis. We detected hyaluronidase activity in the lysate of mycobacteria and showed that it was critical for hyaluronan-dependent extracellular growth. Finally, we showed that L-Ascorbic acid 6-hexadecanoate, a hyaluronidase inhibitor, suppressed growth of mycobacteria in vivo. Taken together, our data show that pathogenic mycobacteria exploit an intrinsic host-protective molecule, hyaluronan, to grow in the respiratory tract and demonstrate the potential usefulness of hyaluronidase inhibitors against mycobacterial diseases.

Transient role of CD4+CD25+ regulatory T cells in mycobacterial infection in mice.

CD4(+)CD25(+) regulatory T (Treg) cells cause immune suppression by inhibiting T cell effector functions and play pivotal roles not only in self-tolerance but also in immune response to parasitic microbial pathogens. Mycobacteria are major parasitic bacterial pathogens, but the role of CD4(+)CD25(+) Treg cells in mycobacterial infection is not yet defined. In this study we found that, at the early stage of infection, depletion of CD25(+) cells reduced both bacterial load and granuloma formation in mice infected with Mycobacterium tuberculosis strains, such as M. tuberculosis Erdman or M. tuberculosis Kurono. However, at a later stage of infection, bacterial burden and histopathology were similar regardless of depletion of CD25(+) cells. Severe combined immunodeficient (SCID) mice reconstituted with CD4(+)CD25(-) T cells alone or a combination of CD4(+)CD25(+) and CD4(+)CD25(-) T cells showed similar bacterial loads and survival kinetics after infection with M. tuberculosis Erdman. Consistent with in vivo data, in vitro studies revealed that mycobacterial antigens, purified protein derivative of tuberculin (PPD), failed to induce the suppressive function of CD4(+)CD25(+) Treg cells to CD4(+)CD25(-) effector T cells, as demonstrated by the lack of response of CD4(+)CD25(+) T cells to PPD, in mice chronically infected with Mycobacterium bovis bacillus Calmette-Guérin and M. tuberculosis. Our data show that CD4(+)CD25(+) Treg cells have a transient effect at the early stage of mycobacterial infection but, contrary to the expectation, have little impact on the overall course of infection.

Pre- and postnatal exposure to low-dose diesel exhaust impairs murine spermatogenesis.

We investigated whether pre- and postnatal low-dose exposure to diesel exhaust (DE) affects male reproductive function in mice. The DE concentration is less than that indicated as the environmental quality standard for suspended particulate matter (SPM) in Japan. ICR mice were exposed prenatally to low-dose diesel exhaust (0.17 mg of DE particles/m³) through the airway for 8 h/day in an exposure chamber from gestational day 2 until the examination. In the DE-exposed groups, normal sperm morphology in the epididymis was reduced (p < 0.01), and seminiferous tubules showed degenerative changes in which the number of Sertoli cells was decreased (p < 0.01). Those changes were observed at 6 and 12 weeks of age. Furthermore, ultrastructural studies revealed an increase in damaged mitochondria in Sertoli cells (p < 0.001) and variform spermatozoa. These results indicate that pre- and postnatal exposure of low-dose DE is detrimental to Sertoli cell function and may cause abnormal spermatozoa.

Nrf2 is closely related to allergic airway inflammatory responses induced by low-dose diesel exhaust particles in mice.

We have recently reported that disruption of nuclear erythroid 2 P45-related factor 2 (Nrf2) enhances susceptibility to airway inflammatory responses induced by low-dose diesel exhaust particles (DEP) in mice. C57BL/6 Nrf2 knockout (Nrf2(-/-)) mice and wild-type (Nrf2(+/+)) mice were further exposed to low-dose DEP for 7h/day, 5 days/week, for a maximum of 8 weeks. After exposure to DEP for 5 weeks, allergic airway inflammation was generated in the mice by intraperitoneal sensitization with OVA followed by intranasal challenge. Nrf2(-/-) mice exposed to relatively low-dose DEP showed significantly increased percentage changes relative to the OVA alone group in terms of airway hyperresponsiveness (AHR) and inflammatory cells, levels of IL-5 and thymus and activation regulated chemokine (TARC) in bronchoalveolar lavage (BAL) fluid than did Nrf2(+/+) mice. Lung tissues of Nrf2(-/-) mice after DEP exposure showed inflammatory cell infiltrates, and increased PAS staining-positive mucus cell hyperplasia. In contrast, the percentage changes relative to the OVA group in the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio in whole blood was higher in Nrf2(+/+) mice than in Nrf2(-/-) mice. By using Nrf2(-/-) mice, it was shown for the first time that relatively low-dose DEP exposure induces oxidant stress, and that host anti-oxidant responses play a key role in the development of DEP-induced exacerbation of allergic airway inflammation.

Identification of Mycobacterium tuberculosis clinical isolates in Bangladesh by a species distinguishable multiplex PCR.

BACKGROUND: Species identification of isolates belonging to the Mycobacterium tuberculosis complex (MTC) seems to be important for the appropriate treatment of patients, since M. bovis is naturally resistant to a first line anti-tuberculosis (TB) drug, pyrazinamide, while most of the other MTC members are susceptible to this antimicrobial agent. A simple and low-cost differentiation method was needed in higher TB burden countries, such as Bangladesh, where the prevalence of M. bovis among people or cattle has not been investigated. METHODS: Genetic regions cfp32, RD9 and RD12 were chosen as targets for a species distinguishable multiplex PCR and the system was evaluated with twenty reference strains of mycobacterial species including non-tubercular mycobacteria (NTM). A total of 350 clinical MTC isolates obtained in Bangladesh were then analyzed with this multiplex PCR. RESULTS: All of the MTC reference strains gave expected banding patterns and no non-specific amplifications were observed in the NTM strains. Out of 350 clinical isolates examined by this method, 347 (99.1%) were positive for all of the cfp32, RD9 and RD12 and determined as M. tuberculosis. Two isolates lacked cfp32 PCR product and one lacked RD12, however, those three samples were further examined and identified as M. tuberculosis by the sequence analyses of hsp65 and gyrB. CONCLUSIONS: The MTC-discrimination multiplex PCR (MTCD-MPCR) developed in this study showed high specificity and was thought to be very useful as a routine test because of its simplicity. In the current survey, all the 350 MTC isolates obtained from Bangladesh TB patients were determined as M. tuberculosis and no other MTC were detected. This result suggested the general TB treatment regimen including pyrazinamide to be the first choice in Bangladesh.

In utero exposure to a low concentration of diesel exhaust affects spontaneous locomotor activity and monoaminergic system in male mice.

BACKGROUND: Epidemiological studies have suggested that suspended particulate matter (SPM) causes detrimental health effects such as respiratory and cardiovascular diseases, and that diesel exhaust particles from automobiles is a major contributor to SPM. It has been reported that neonatal and adult exposure to diesel exhaust damages the central nervous system (CNS) and induces behavioral alteration. Recently, we have focused on the effects of prenatal exposure to diesel exhaust on the CNS. In this study, we examined the effects of prenatal exposure to low concentration of diesel exhaust on behaviour and the monoaminergic neuron system. Spontaneous locomotor activity (SLA) and monoamine levels in the CNS were assessed. METHODS: Mice were exposed prenatally to a low concentration of diesel exhaust (171 microg DEP/m(3)) for 8 hours/day on gestational days 2-16. SLA was assessed for 3 days in 4-week-old mice by analysis of the release of temperature-associated infrared rays. At 5 weeks of age, the mice were sacrificed and the brains were used for analysis by high-performance liquid chromatography (HPLC). RESULTS AND DISCUSSION: Mice exposed to a low concentration of diesel exhaust showed decreased SLA in the first 60 minutes of exposure. Over the entire test period, the mice exposed prenatally to diesel exhaust showed decreased daily SLA compared to that in control mice, and the SLA in each 3 hour period was decreased when the lights were turned on. Neurotransmitter levels, including dopamine and noradrenaline, were increased in the prefrontal cortex (PFC) in the exposure group compared to the control group. The metabolites of dopamine and noradrenaline also increased in the PFC. Neurotransmitter turnover, an index of neuronal activity, of dopamine and noradrenaline was decreased in various regions of the CNS, including the striatum, in the exposure group. The serum corticosterone level was not different between groups. The data suggest that decreased SLA in mice exposed prenatally to diesel exhaust is due to facilitated release of dopamine in the PFC. CONCLUSIONS: These results indicate that exposure of mice in utero to a low concentration of diesel exhaust decreases SLA and alters the neurochemical monoamine metabolism of several regions of the brain.

Development of new anti-tuberculosis drug candidates.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a tenacious and remarkably successful pathogen that has latently infected one third of the world's population, according to the World Health Organization (WHO) statistics. It is anticipated that 10% of these infected individuals will develop active tuberculosis at some point in their lifetime. The long-term use of the current drug regimen, the emergence of drug-resistant strains, and HIV co-infection have resulted in a resurgence of research efforts to address the urgent need for new anti-tuberculosis drugs. A number of potential candidate drugs with novel modes of action have entered clinical trials in recent years, and these are likely to be effective against anti-tuberculosis drug-resistant strains. They include neuroquinolone derivatives, a modified ethambutol, nitro-imidazole groups and so on. This mini-review summarizes the latest information about eight new anti-tuberculosis drug candidates and describes their activities, pharmacokinetics, mechanisms of action, and mechanisms of drug-resistance induced by these drug candidates.

Effect of prenatal exposure to diesel exhaust on dopaminergic system in mice.

Diesel exhaust (DE) is composed of particles and gaseous compounds. It has been reported that DE causes pulmonary and cardiovascular disease. We have previously reported that fetal exposure to DE had deleterious effects to the reproductive system of mice offspring. However, there is still little known about the effects of prenatal exposure to DE to the central nervous system (CNS). In the present study, we found that prenatal exposure to DE induced reduction of locomotion, furthermore, dopamine (DA) turnover was significantly decreased in the striatum and nucleus accumbens. These results suggest that prenatal exposure to DE has an effect on the CNS. Hypolocomotion could be due to a decrease in DA turnover associated with DA nervous system abnormality. The present study provides the possibility that maternally inhaled DE might influence the development of central dopaminergic system and result in behavior disorder.

Tuberculosis complicated by diabetes mellitus at shanghai pulmonary hospital, china.

An association between diabetes mellitus (DM) and tuberculosis (TB) has been implied for a long time. We previously reported that KDP type 1 diabetic rats and GK type 2 diabetic rats are highly susceptible to Mycobacterium tuberculosis infection. As a next step, we conducted a retrospective analysis of 2,141 patients with pulmonary TB newly diagnosed during the period from 2008 to 2009 to evaluate the influence of DM on the drug response rate and the long-term relapse rate of TB. There were 203 DM patients with TB (type 1 DM, 7 [3.4%]; type 2 DM, 196 [96.6%]). The TB relapse rate (2 years after discharge) was higher in DM patients than in non-diabetic patients (20% versus 5.3%). The frequency of multidrug-resistant-TB among DM patients with TB was higher than that among TB patients (17.7% versus 8.4%, P<0.01). These results suggest that the period of TB treatment should be prolonged, and that in the meantime the blood glucose level should be maintained within a reference value range.

Establishment of a guinea pig model of latent tuberculosis with GFP-introduced Mycobacterium tuberculosis.

There exists latent tuberculosis, in which small numbers of tubercle bacilli remain viable in the host without visible granulomatous lesions. As few data exist on the mechanisms of latent tuberculosis, it is important to examine latent tuberculosis in terms of pathogenesis and efficacy of chemotherapy. As a first step, we used green fluorescent protein (GFP)-introduced H37Rv Mycobacterium tuberculosis to establish latent tuberculosis in the guinea pig that provides one of the best animal models of tuberculosis. We inoculated the guinea pigs subcutaneously with 100 or 1,000 colony-forming unit (CFU) of tubercle bacilli. During the 300-day follow-up period after infection, there were no clinical signs of disease, suggesting a lack of visible granulomatous lesions. In fact, upon necropsy, no macroscopic tuberculous lesions were recognized, but histopathological examination of the lung, spleen and liver revealed microgranulomas consisting of epithelioid macrophages and lymphocytes without central necrosis. Importantly, photon imaging visualized granulomatous lesions corresponding to these histologically apparent microgranulomas. Tuberculin skin testing of infected guinea pigs showed strong positivity (> or = 10 mm induration) until the end of the experiments. Real-time PCR analysis showed a significant increase in the expression levels of interferon-gamma, tumor necrosis factor-alpha, interleukin-12, and inducible nitric oxide synthase mRNAs in infected lung tissues after 300 days (P < 0.01). As human samples are hardly available to study latent tuberculosis, our guinea pig model would be useful for examining the pathogenesis and molecular mechanisms of latent tuberculosis as well as for monitoring the results of chemotherapy with green fluorescence emission of tubercle bacilli.

The effects of oxidative stress induced by prolonged low-dose diesel exhaust particle exposure on the generation of allergic airway inflammation differ between BALB/c and C57BL/6 mice.

We have recently reported that airway inflammatory responses to the oxidative stress induced by prolonged low-dose diesel exhaust particle (DEP) exposure differ markedly between BALB/c and C57BL/6 mice. In the present study, the effects of genetic differences in the response to prolonged low-dose DEP exposure on the generation of ovalbumin-induced allergic airway inflammation were further explored using the same mouse strains. In BALB/c mice, eosinophils and mucous goblet cells in histopathological pulmonary specimens increased significantly after DEP exposure, and were more marked than in C57BL/6 mice. Interleukin (IL)-5 and IL-13 levels in bronchoalveolar lavage (BAL) fluid were increased significantly by DEP exposure only in BALB/c mice. The DEP-induced increases in peribronchial eosinophils and mucous goblet cells in the lung tissues, and of IL-5 and IL-13 in the BAL fluid, were significantly attenuated by the antioxidant N-acetylcysteine. Thus, the effects of prolonged low-dose DEP exposure on the generation of allergic airway inflammation differed markedly between the mouse strains. These differences may be caused by different antioxidant responses to the oxidative stress induced by DEP exposure. Our results contribute more information to the search for genetic susceptibility factors in the response to DEP, and may thus assist in the discovery of new biomarkers for DEP-related disease.

Aggravating effect of natural sand dust on male reproductive function in mice.

PURPOSE: Although adverse health effects of environment (such as cadmium, pesticides, diesel exhaust, etc.) on the male reproductive system have been suggested, there is little experimental evidence of such an effect of atmospheric sand dust. In the present study, the effects of sand dust (mineral particles) were investigated on the male reproductive system of mice. METHODS: Two types of sand dusts (Asian sand dust and Arizona sand dust) were intratracheally administered (0.1 mg/mouse 4 times every other week) to ICR male mice and then male reproductive organ weight, daily sperm production (DSP), histological analysis and serum testosterone level were measured. RESULTS: Histological examination showed that interstitial edema was produced by both sand dust types, and partial vacuolation of the seminiferous tubules was detected in the exposed mice. Moreover, exposure to these natural sand dusts significantly decreased DSP. On the other hand, there was no significant differences in serum testosterone concentration. CONCLUSIONS: These results suggest that natural sand dust-exposure produced adverse effects on mouse male reproductive function.

Why does tuberculosis lead to specific inflammation?

When Mycobacterium tuberculosis infects humans, about 20% of those infected actually develop tuberculosis (TB). In Japan, the incidence of TB in 2008 was 24,760 cases (19.4/100,000 persons) and the rate has been decreasing gradually, but is still higher than in the USA, Holland, and Belgium, for example. Histologically, tuberculosis displays exudative inflammation, proliferative inflammation and productive inflammation depending on the time course. In productive inflammation, granulomatous lesions with necrotic centers are formed. The typical granulomas consist of epithelioid macrophages, Langhans' multinucleated giant cells, lymphocytes and fibroblasts, and the process of their formation involves many cytokines, chemokines and transcription factors. These findings have been derived primarily from animal experiments utilizing an airborne infection apparatus. The conditions for airborne infection have been described in detail elsewhere. This mini-review focuses on what has been found through animal experiments, and also indicates areas for which data are not currently available.

Disruption of Nrf2 enhances susceptibility to airway inflammatory responses induced by low-dose diesel exhaust particles in mice.

To test our hypothesis that diesel exhaust particle (DEP)-induced oxidative stress and host antioxidant responses play a key role in the development of DEP-induced airway inflammatory diseases, C57BL/6 nuclear erythroid 2 P45-related factor 2 (Nrf2) knockout (Nrf2(-/-)) and wild-type mice were exposed to low-dose DEP for 7 h/day, 5 days/week, for 8 weeks. Nrf2(-/-) mice exposed to low-dose DEP showed significantly increased airway hyperresponsiveness and counts of lymphocytes and eosinophils, together with increased concentrations of IL-12 and IL-13, and thymus and activation-regulated chemokine (TARC), in BAL fluid than wild-type mice. In contrast, expression of antioxidant enzyme genes was significantly higher in wild-type mice than in Nrf2(-/-) mice. We have first demonstrated that disruption of Nrf2 enhances susceptibility to airway inflammatory responses induced by inhalation of low-dose DEP in mice. These results strongly suggest that DEP-induced oxidative stress and host antioxidant responses play some role in the development of DEP-induced airway inflammation.

Promising loci of variable numbers of tandem repeats for typing Beijing family Mycobacterium tuberculosis.

We analysed the genotypes of 325 Mycobacterium tuberculosis clinical isolates obtained during 2002 throughout Japan. The genotyping methods included insertion sequence IS6110 RFLP, spoligotyping and variable number of tandem repeat (VNTR) analyses. Clustered isolates revealed by IS6110 RFLP analysis accounted for 18.5 % (60/325) of the isolates. Beijing genotype tuberculosis (TB) accounted for 73.8 % (240/325) of the isolates. Using VNTR, we analysed 35 loci, including 12 standard mycobacterial interspersed repetitive units and 4 exact tandem repeats. The discriminatory power of these 16 loci was low. Using VNTR analyses of the 35 loci, 12 loci (VNTRs 0424, 0960, 1955, 2074, 2163b, 2372, 2996, 3155, 3192, 3336, 4052 and 4156) were selected for the genotyping of Beijing genotype strains. Comparison of the discriminatory power of the 12-locus VNTR [Japan Anti-Tuberculosis Association (JATA)] to that of the 15-locus and 24-locus VNTRs proposed by Supply et al. (2006) showed that our established VNTR system was superior to the reported 15-locus VNTR and had almost equal discriminatory power to the 24-locus VNTR. This 12-locus VNTR (JATA) can therefore be used for TB genotyping in areas where Beijing family strains are dominant.

Histopathological classification of systemic Mycobacterium avium complex infections in slaughtered domestic pigs.

The aim of this study was to classify the histopathological features of pigs infected with Mycobacterium avium complex (MAC). We used slaughtered pig organs systemically infected with MAC. The results showed granulomatous lesions which were observed predominantly in the digestive organs and regional lymph nodes rather than respiratory organs. The histological picture showed a wide range of granulomatous stages from exudative to fibrotic reactions to the MAC infection. Eosinophils and giant cells were characteristically observed in the exudative reactions. The histopathological type in primary focus tended to be maintained in the respective organs. Most strains with the same genotype showed pathogenicity for guinea pigs irrespective of the type of granuloma. Although these findings suggest that different stages of a granulomatous lesion originating from the same causative agent might influence histological patterns, other possibilities such as the hereditary background of the host, or the effects of viral infections should be considered.

Detrimental effects of prenatal exposure to filtered diesel exhaust on mouse spermatogenesis.

We recently showed that prenatal exposure to diesel exhaust (DE) disrupts spermatogenesis in mouse offspring. This study was undertaken to determine whether filtered DE in which 99.97% of diesel exhaust particles >0.3 microm in diameter were removed affects spermatogenesis in growing mice. After prenatal exposure to filtered DE for 2-16 days postcoitum, we examined daily sperm production (DSP), testicular histology, serum testosterone levels and mRNA expression of hormone synthesis process-related factors. In the filtered DE exposed group, DSP was markedly reduced at 12 weeks compared with the control group; clean air exposed group. Histological examination showed multinucleated giant cells and partial vacuolation in the seminiferous tubules of the exposed group. Testosterone was elevated significantly at 5 weeks. Moreover, luteinizing hormone receptor mRNA at 5 and 12 weeks, 17alpha-hydroxylase/C17-20-lyase and 17beta-hydroxysteroid dehydrogenase mRNAs at 12 weeks were significantly elevated. These results suggest that filtered DE retains its toxic effects on the male reproductive system following prenatal exposure.

Higher susceptibility of type 1 diabetic rats to Mycobacterium tuberculosis infection.

An association between diabetes mellitus and tuberculosis has been implicated for a long time. We have previously reported that Goto Kakizaki type 2 diabetic rats are highly susceptible to Mycobacterium (M.) tuberculosis infection. As a next step, we attempted to clarify whether type 1 diabetic rats are more susceptible to M. tuberculosis than non-diabetic wild-type (WT) rats. Here, we used the Komeda diabetes-prone (KDP) rat, as a model of type 1 diabetes mellitus. The infected KDP rats developed large granulomas without central necrosis in their lungs, liver or spleen. This was consistent with a significant increase in the number of colony-forming units (cfu) of M. tuberculosis in the lungs and spleen (p < 0.01). Insulin treatment resulted in significant reduction of tubercle bacilli in the infected KDP rats (p < 0.01). Pulmonary levels of interferon-gamma, tumor necrosis factor-alpha and interleukin-1beta mRNAs were higher in the infected diabetic rats than in WT rats. Alveolar macrophages from KDP rats were not fully activated by M. tuberculosis infection because the macrophages did not secrete nitric oxide (NO) that can kill M. tuberculosis (p < 0.01), but no significant difference in phagocytosis of tubercle bacilli by alveolar macrophages was observed between KDP and WT rats. Taken together, our findings indicate that type 1 diabetic rats are more susceptible to M. tuberculosis that WT rats.

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

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