[show abstract][hide abstract] ABSTRACT: The rapid identification of acid-fast bacilli recovered from patient specimens as Mycobacterium tuberculosis complex (MTC) is critically important for accurate diagnosis and treatment. A thin-layer immunochromatographic (TLC) assay using anti-MPB64 or anti-MPT64 monoclonal antibodies was developed to discriminate between MTC and non-tuberculosis mycobacteria (NTM). Capilia TB-Neo, which is the improved version of Capilia TB, is recently developed and needs to be evaluated.
Capilia TB-Neo was evaluated by using reference strains including 96 Mycobacterium species (4 MTC and 92 NTM) and 3 other bacterial genera, and clinical isolates (500 MTC and 90 NTM isolates). M. tuberculosis isolates tested negative by Capilia TB-Neo were sequenced for mpt64 gene.
Capilia TB-Neo showed 100% agreement to a subset of reference strains. Non-specific reaction to M. marinum was not observed. The sensitivity and specificity of Capilia TB-Neo to the clinical isolates were 99.4% (99.6% for M. tuberculosis, excluding M. bovis BCG) for clinical MTC isolates and 100% for NTM isolates tested, respectively. Two M. tuberculosis isolates tested negative by Capilia TB-Neo: one harbored a 63-bp deletion in the mpt64 gene and the other possessed a 3,659-bp deletion from Rv1977 to Rv1981c, a region including the entire mpt64 gene.
Capilia TB-Neo is a simple, rapid and highly sensitive test for identifying MTC, and showed better specificity than Capilia TB. However, Capilia TB-Neo still showed false-negative results with mpt64 mutations. The limitation should be recognized for clinical use.
[show abstract][hide abstract] ABSTRACT: Mycobacteria consist of 2 large groups: one is the tuberculosis complex, and the other is nontuberculous mycobacterium (NTM). Most of the NTM are generally non-virulent bacteria, but some NTMs have pathogenicity to humans. There are many reports of nosocomial infection cases caused by common bacteria such as multidrug-resistant Pseudomonas aeruginosa. Also, some cases of in-hospital infection due to NTM were reported. Unlike common bacteria, detection of mycobacteria is affected by various factors, such as stainability, time for colony forming, temperature and nutrition Mycobacterium chelonae chemovar niacinogenes was isolated from 5 patients in 73 nosocomial infection cases (60 patients and 13 suspected cases) at a certain hospital during the period from March 2007 until January 2009. One of the reasons for the expansion of infection and difficulty in identification of the bacteria was the properties of this mycobacterium. This bacterium was very faintly stained with Gram-staining. Therefore, this mycobacterium could only be detected at a hospital when Ziehl-Neelsen stain and the cultivation at 28 degrees C for more than 5 days were performed. MICs for Cefmenoxime and Tosufloxacin of the isolates were more than 128 microg/mL. The isolates and type strasin of M. chelonae chemovar niacinogenes were also resistant to other drugs.
Kansenshogaku zasshi. The Journal of the Japanese Association for Infectious Diseases 07/2013; 87(4):424-30.
[show abstract][hide abstract] ABSTRACT: Two new mycolactones, mycolactones S1 and S2, were isolated from culture agar of Mycobacterium ulcerans subsp. shinshuense. Their structures were established in a three-step procedure: (1) probable structures were speculated from MS analysis; (2) candidates were synthesized; (3) HPLC profiles were established for identification of the natural products. Newly isolated mycolactones correspond to the "oxidized forms" of mycolactone A/B, the causative toxin of Buruli ulcer, isolated from Mycobacterium ulcerans.
[show abstract][hide abstract] ABSTRACT: Background
Conventional biochemical tests are the standard for the identification of Mycobacterium species, but molecular identifications are becoming more prevalent. The rpoB gene encodes the β-subunit of RNA polymerase and is utilized for the identification of Mycobacterium species. In the present study, a stepwise Mycobacterium species identification algorithm using the 16S rRNA encoding gene and rpoB analysis was evaluated for its effectiveness.MethodsA total of 172 clinical Mycobacterium isolates were tested, and concordant results were obtained with 108 strains by using the conventional method and molecular methods (AccuProbe or DDH method).ResultsIn these 108 strains, 4 strains were identified by 16S rRNA gene analysis, but rpoB indicated no identical Mycobacterium species with more than 99% similarity.The remaining 64 strains were not identified by conventional method and commercial kits. Forty-two showed concordant results with 16S rRNA and rpoB analysis, and 13 strains were identified by 16S rRNA gene analysis although rpoB indicated no identical Mycobacterium species. On the other hand, 4 strains included 2 strains of Gordona and 2 strains of M. celatum type II which were identified by rpoB but not by 16S rRNA gene analysis. Finally, 5 strains could not be identified by analysis of either gene. The rpoB analysis can differentiate M. kansasii from M. gastri; M. malmoense from M. szulgai; M. abscessus from M. chelonae; M. peregrinum from M. septicum; M. porcinum from M. fortuitum; and M. farucinogense from M. senegalense—pairs that are not differentiated by 16S rRNA analysis. Additionally, Nocardia asteroids, Rhodococcus equi, Gordona aichiense, G. aurantiaca, G. bronchialis and G. terrae are able to be analyzed by using rpoB.Conclusions
The 16S rRNA gene identification is a rapid and prevalent method but still has some limitations. Therefore, the stepwise combination of rpoB with 16S rRNA gene analysis is an effective system for the identification of Mycobacterium species.
International Journal of Mycobacteriology. 03/2012; 1(1):21–28.
[show abstract][hide abstract] ABSTRACT: Immune reconstitution inflammatory syndrome (IRIS) caused by mycobacterium in patients with AIDS is often experienced in clinical practice. There is, however, a paucity of data documenting the histopathological findings and the pathogenesis. We determined the immunopathological characteristics of IRIS associated with Mycobacterium parascrofulaceum infection in an AIDS patient. A patient presented with pulmonary lymphadenitis and involvement of the pulmonary lingular segment. Portions of the involved lymph nodes and lung were excised, and the immunological properties were analyzed by immunohistochemical assays. The histological characteristics of lymph nodes showed a caseous necrosis. Histopathologically, the pulmonary lesion was composed of exudative and proliferative lesions. CD4(+), CD8(+), CD57(+), and CD25(+)/FoxP3(+) cells were observed in both types of lesions. Clusters of CD20(+) cells and GATA3(+) cells were predominantly observed in exudative lesions, while T-bet(+) cells were dominant in proliferative lesions. ROR-γ(+) cells were also observed in exudative lesions. These results indicate that the cellular immunity to mycobacteria was recovering in the lung tissue. In M. parascrofulaceum pulmonary infection, the exudative lesion had characteristics of Th2 and Th17-type immunities. In contrast, the proliferative lesion had characteristics of Th-1 type immunity. Our data provide the first evidence to reveal the status of the axis of distinctive immunity in the process of granuloma formation caused by a mycobacterium-related infection.
Pathology - Research and Practice 03/2011; 207(4):262-70. · 1.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: The DNA sequencing analyses of the 16S rRNA gene, rpoB and hsp65 were conducted to characterize six strains that had been identified as Mycobacterium xenopi by DNA-DNA hybridization (DDH) for past ten years in our hospital. The results revealed Mycobacterium heckeshornense infection in one of the six cases. A 47-year-old man, who had been treated for pneumonia, had pulmonary nontuberculous mycobacterial disease. The sputa from the patient were culture positive for mycobacterium in three times. And it was diagnosed as M. xenopiby DDH method. Chest X-ray showed fibrocavitary lesion in right upper lobe was successfully treated with clarithromycin for four weeks.
Internal Medicine 01/2011; 50(11):1251-3. · 0.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: We analyzed the clinical characteristics of extensively-drug resistant tuberculosis (XDR-TB).
Thirteen cases diagnosed with XDR-TB encountered in our hospital over the last ten years were enrolled in our study.
The patients included 9 males and 4 females. The mean age was 49.1 years old in males and 42.0 years old in females. Eight patients were Japanese and 5 were foreigners (Chinese, 3; Korean, 1; Nepali, 1). Nine cases had a smoking history and 6 had underlying diseases, including 1 with bacterial pneumonia, 3 with diabetes mellitus, 1 with chronic renal failure, and 1 with collagen vascular disease receiving immunosuppressive treatment. All 13 cases had been diagnosed at other hospitals. The mean period from TB diagnosis to XDR-TB diagnosis was 56.8 months, and the mean period from TB diagnosis to referral to our hospital was 81.6 months. Among the 13 cases, 3 had no drug sensitivity, 1 was sensitive to only 1 drug, 2 were sensitive to 2 drugs, 6 were sensitive to 3 drugs, and 1 was sensitive to 4 drugs. Nine of the 13 cases had surgical treatment. Six cases, all of whom had surgical treatment, showed negative conversion in sputum examinations. Three patients died, including two who had surgical treatment. Among the 3 cases with no drug sensitivity, 1 was cured after surgical treatment. Another case had been working in the same hospital with two other MDR-TB cases. Two of the three had the same RFLP pattern.
XDR-TB and MDR-TB are man-made diseases. We need to take measures not to create more XDR strains and induce more MDR-TB cases.
[show abstract][hide abstract] ABSTRACT: Saito et al. isolated novel mycobacterium strains from the sputum of 12 patients with pulmonary disease. They reported, that the strains were clearly different from Mycobacterium tuberculosis (TB) in cultural, biochemical and immunological properties, despite the high homology (99.1%) of the 16S rRNA gene sequence between the two. Recently, we isolated four strains having similar properties as the above strains among mycobacterial strains that were sent to the Research Institute of Tuberculosis for identification. We examined these isolates using commercial systems for identification of mycobacteria.
Four strains of the unidentified mycobacteria were used in this study.
Tests used in the study included cultural on solid media, biochemical characteristics, DNA sequence analyses of 16S rRNA and rpoB genes, TRC, COBAS AMPLICOR, COBAS TaqMan, MTD, DDH, Accu-Probe, Capilia TB, and a drug susceptibility tests.
After three weeks of culture, smooth and non-photochromogenic colonies were formed. The niacin accumulation test was negative. The homologies of DNA sequence between the new strains and M. tuberculosis for 16S rRNA and rpoB genes were 97.8% and 90.2%, respectively. The tests with TRC and MTD kits were positive, whereas the tests with AMPLICOR, TaqMan, Accu-Probe and Capilia TB kits were negative. The organism was not identified with the DDH system.
[show abstract][hide abstract] ABSTRACT: Recent genetic studies have revealed that several epidemiological factors affect Mycobacterium avium complex (MAC) infection in pig populations. However, mechanisms underlying the spread of MAC infection among hog farms have not been clarified. In consideration of this situation, we cross-sectionally investigated the mechanisms underlying the spread of MAC on the island of Okinawa. Pigs slaughtered (n = 706,763) and 331 hog farms on Okinawa were surveyed during the years 2002–2004. Two outbreaks of MAC infection were occurred in several farms during survey period. Bacteria were isolated from randomly selected pigs and genotype of isolates was determined by using genetic finger printing methods with the insertion sequence (IS) 1245 restriction fragment length polymorphism (RFLP). Most isolates had large numbers of IS1245 copies, while strains with low copy numbers of IS1245 and isolates without IS1245 were seen in few farms. MACs strains were repeatedly isolated from pigs of the affected farms during the survey period. Those farms with an identical pig rearing systems showed synchronic changes in the prevalence of MAC infection. An industrial farm without an outbreak had an independent pig flow, but maintained distinct MAC strains. Multivariate analysis did not reveal independent factors for the prevalence of the MAC infection. These findings suggest that there were three clusters distinguished genetically in the main island of Okinawa, which were potentially spread by common pig flow. However, the outbreaks occurred because of unspecified conditions on each farm environment.RésuméDe récentes études en génétique ont dévoilé que plusieurs facteurs épidémiologiques ont des répercussions néfastes sur le complexe Mycobacterium avium (MAC) des porcs, ce qui les rend malades. Cependant, les mécanismes sous-jacents à la diffusion de l’infection du MAC dans les élevages de porcs n’ont pas été clairement identifiés. Par conséquent, nous avons examiné les mécanismes latents à la propagation du MAC sur l’île d’Okinawa en découpant la population totale des porcs en groupes d’animaux présentant les mêmes caractéristiques. Notre étude recense donc 706 763 bêtes réparties dans 331 fermes. Sur toute la période de nos travaux, le taux moyen des infections du MAC atteind au maximum 9,2% des animaux. Les fermes présentant des systèmes d’élevages identiques ont montré des modifications simultanées dans la fréquence des infections, et les organismes aux mêmes caractéristiques génétiques ont été séparés des porcs de ces fermes.Une ferme d’élevage en batterie dont les animaux proviennent d’un marché indépendant ne présente aucun pic d’infection, et garde des distinctions génétiques au niveau du MAC. Ces conclusions suggèrent qu’il y avait trois groupes distingués génétiquement dans l’île principale d’Okinawa qui s’est été étendu potentiellement par courant du cochon commun. Cependant, les premières manifestations se sont produites à cause de conditions non spécifiées sur chaque environnement de ferme.
Comparative Immunology, Microbiology and Infectious Diseases. 01/2010;
[show abstract][hide abstract] ABSTRACT: Nontuberculous mycobacterial (NTM) infection in HIV (human immunodeficiency virus)-infected patients who have started highly active antiretroviral therapy (HAART) is well known to be one scenario of immune reconstitution inflammatory syndrome (IRIS). We encountered the first case in Japan of an HIV-infected patient with pulmonary Mycobacterium parascrofulaceum infection as IRIS. A 34 year-old man with acquired immunodeficiency syndrome (AIDS) was receiving highly active antiretroviral therapy. Lymphadenopathy was observed at the left pulmonary hilum. IRIS was suspected and thoracoscopic surgery was performed to diagnose the cause of lymphadenopathy. Granulomas were observed histologically, and M. parascrofulaceum was cultured. This organism was susceptible to Clarithromycin, rifampicin and levofloxacin. After the operation and without treatment, recurrence of M. parascrofulaceum infection was not observed. M. parascrofulaceum was isolated from several clinical specimens for the first time in 2004. To date, only five cases have been reported.
Internal Medicine 01/2010; 49(16):1817-21. · 0.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mycobacterium porcinum has been successfully isolated from the patient with abnormal signal transduction pathway of IL12/IFN-gamma. The properties of each bacterium were determined by conventional identification methods, DNA sequencing analysis and MIC assay.
M. porcinum was isolated 7 times from 1996 to 2007 from cervical lymph node, axillary lymph nodes, inguinal lymph node, brachial lymph node and site of a tumor of the patient. In another occasion, mycobacteria were isolated from lavage fluid of the endoscope in routine inspection. Using these mycobacteria, M. porcinum (ATCC33776) and M. fortuitum (ATCC6841), the conventional identification method and MIC assay were carried out. For analyses of the DNA sequencing (rpoB, dnaJ and hsp65), the ATCC type strain of mycobacteria (11 strains) which are closely related to M. porcinum were also used.
DNA sequencing analyses of the 7 samples isolated from the patient, were concurrently identical in 3 different genes. Drug susceptibility test showed that 7 isolates had no marked change. In conventional identification analyses, M. porcinum (ATCC33776), M. fortuitum (ATCC6841), and M. porcinum that were isolated in 1996, were able to grow at 42 degrees C. However, 6 isolates that were isolated after 1999, did not grow at 42 degrees C. The colony detectable days of these 7 strains changed from 3 to 7. Over the time, the morphology of each colony changed from smooth to rough. Though the initial isolate had the ability to utilize mannitol, the later 4 isolates had no such ability.
[show abstract][hide abstract] ABSTRACT: The bacilli which were isolated from a patient suspected of the mixed infections with Mycobacterium avium and Mycobacterium intracellulare, were analyzed. The genotypes of M. avium in the sedimented fractions of treated sputum and in some colonies isolated from Ogawa medium were compared by the Variable Numbers of Tandem Repeats (VNTR).
A woman, aged 57. Mycobacterial species isolated from some colonies by culture in 2004 and 2006 and from the treated sputum in 2006, were determined by DNA sequencing analysis of the 16S rRNA gene. Also, by using VNTR, the genotype of mycobacteria was analyzed. [Results] (1) The colony isolated from Ogawa medium in 2004 was monoclonal M. avium. (2) By VNTR analyses of specimens in 2006, multiple acid-fast bacteria were found in the sputum sediment and in isolated bacteria from Ogawa medium. (3) By analyses of 16S rRNA DNA sequence, M. avium and M. intracellulare were found in the colonies isolated from the sputum sediment and the Ogawa medium in 2006. (4) The same VNTR patterns were obtained in M. avium in 2004 and 2006 when single colony was analyzed. (5) From the showerhead and culvert of the bathroom in the patient's house, M. avium was not detected.
By VNTR analyses, it was considered that the mixed infections of M. avium and M. intracellulare had been generated during treatment in this case. Therefore, in the case of suspected complex infection, VNTR analysis would be a useful genotyping method in M. avium complex infection.
[show abstract][hide abstract] ABSTRACT: The Invader assay was developed to identify 23 mycobacterial species using probes derived from the species-specific region of the 16S rRNA gene and the 16S-23S rRNA internal transcribed spacer 1 (ITS-1) region, with minor modifications of our previous study. In the present study, we compared the identification capability between the Invader assay and DNA-DNA hybridization (DDH) method. DDH is commonly used to identify non-tuberculosis mycobacterium in Japan and 636 clinical mycobacterial strains cultured on Ogawa slants were tested.
The Invader assay could identify 615 (96.7%) of the 636 strains. The results contained 14 M.lentiflavum, 3 M. parascrofulaceum and 1 M. intermedium, which were undetectable with DDH method. On the other hand, DDH method could identify 580 (91.2%) strains with duplicate assay. Of 628 strains except 8 strains identified as a few species by Invader assay, 551 (87.7%) strains were identified as the same species by two methods. Discordant results were mainly recognized for the identification of M. gordonae, M. avium, M. lentiflavum and M. intracellurare. The results of other methods targeting 16S rRNA indicated correctness of the Invader assay.
These results indicate that Invader assay could identify more correctly than DDH method and could identify about 97% of clinically important mycobacterium.
[show abstract][hide abstract] ABSTRACT: To observe the frequency of MDR-TB/XDR-TB strains isolated from chronic pulmonary tuberculosis patients in Japan.
Ad hoc National Tuberculosis Survey 2000 on frequency of MDR-TB and XDR-TB strains.
Four hundred and thirty four clinical isolates were collected by the Ad hoc National Tuberculosis Survey 2000, the drug susceptibility testings (proportion method, MGIT Middlebrook, and BrothMIC NTM) were conducted on these strains. These clinical isolates were obtained from patients registered at Health Centers in Japan by the end of 1999 who were culture-positive in 1999 and were registered before January 1st, 1998. The isolates used in this study were selected from patients who were culture-positive at shortest 2 years after the registration.
The clinical isolates resistant to both INH and RFP were 321 out of 434 (74.0%). The 180 MDR-resistant clinical isolates were also resistant to levofloxacin and amikacin and/or kanamycin. These phenotypes are XDR-TB. No previously registered cases were 165, and previously registered cases were 143 and unknown cases were 13 out of 321 MDR-TB. In 180 XDR-TB cases, no previously registered cases were 95, previously registered cases were 78 and unknown cases were 7. In no previously registered cases, more than 50% cases started treatment in 1990s. Approximately 50% of previously registered patients started treatment in 1960s and 1970s.
We performed drug susceptibility testing for 434 clinical isolates which were culture-positive at shortest 2 years after registration. No. of MDR-TB patients was 321 and that of XDR-TB patients was 180. The treatment outcome of these patients have to be followed up carefully at Health Centers. The frequency of amikacin resistance was relatively high. This may be due to either common use of amikacin or cross-resistance against streptomycin and kanamycin.
[show abstract][hide abstract] ABSTRACT: Comparison of usefulness of IS1245 RFLP and VNTR in M. avium genotyping.
Thirty-six cases (55 strains) from sputum and BALF and twelve cases (29 strains) isolated from blood of HIV-infected patients were used. VNTR and RFLP using IS1245 were performed.
Multiple samples were taken from 16 patients and 52 clinical isolates were used for VNTR and RFLP for comparison. (1) VNTR and RFLP results were identical in 12 out of 16 cases whose samples were collected several times. (2) Eight isolates were obtained from one patient. In this eight isolates, there were the cases of M. avium polyclonal infection and of mixed infection with M. intracellulare. VNTR patterns were two types and RFLP were 5 kinds of different in this case. (3) VNTR patterns of six isolates from one HIV-infected patient were identical, but there were three variations in RFLP patterns. There were three cases of mixed infections with M. tuberculosis or M. intracellulare, and six strains polyclonal infection of M. avium (7.1 %) in 84 isolates. These 6 clinical isolates were derived from sputum or BALF (5 strains) and HIV-infected blood (one strain). VNTR patterns were similar in four pairs (9 strains) who did not contact closely, but they were distinguished clearly by RFLP. Seventeen strains had three or less IS1245-related bands in RFLP analyses of 89 strains.
As there is a possibility of polyclonal infection with M. avium and mixed infection with other species, the single clonal infection should be confirmed first by VNTR. When single colony was obtained, VNTR and RFLP were performed for genotyping of M. avium. Furthermore, strains with less bands by RFLP should be carefully judged in terms of both VNTR and RFLP. It is recommended that the specimens should be collected from each patient several times.
[show abstract][hide abstract] ABSTRACT: A 36 year-old female was pointed out of pulmonary abnormal shadows in the annual chest survey. Chest radiograph and computed tomography (CT) disclosed bilateral diffuse infiltrative shadows and tree-in-bud appearance in the right upper lung field and the left lingula. A sputum smear for acid-fast bacilli was negative. Histopathologically, the transbronchial lung biopsy specimen revealed non-caseous epithelioid granulomas with numerous giant cells. Acid-fast bacilli were cultured from her sputum, however, nontuberculous mycobacteria was not detected by DNA-DNA hybridization method. Mycobacterium mageritense was identified by 16S ribosomal RNA sequencing with 100% matching. The isolated colony of M. mageritense was resistant to nine anti-tuberculous drugs. Follow-up chest CT scan showed a gradual decrease of infiltrative shadows without therapy. To the best of our knowledge, M. mageritense infections are rare, and this is the first case report of pulmonary infection in the literature. We conclude that the pulmonary infection of M. mageritense is one of causes of granuloma formation, and in some case it is difficult to differentiate clinically from sarcoidosis.
[show abstract][hide abstract] 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.
Journal of Clinical Microbiology 01/2007; 44(12):4566-8. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: China is regarded by the World Health Organization as a major hot-spot region for Mycobacterium tuberculosis infection. Streptomycin has been deployed in China for over 50 years and is still widely used for tuberculosis treatment. We have developed a denaturing HPLC (DHPLC) method for detecting various gene mutations conferring drug resistance in M. tuberculosis. The present study focused on rpsL and rrs mutation analysis. Two hundred and fifteen M. tuberculosis clinical isolates (115 proved to be streptomycin-resistant and 100 susceptible by a routine proportional method) from China were tested to determine the streptomycin minimal inhibitory concentration (MIC), and subjected to DHPLC and concurrent DNA sequencing to determine rpsL and rrs mutations. The results showed that 85.2% (98/115) of streptomycin-resistant isolates harbored rpsL or rrs mutation, while rpsL mutation (76.5%, 88/115) dominated. MIC of 98 mutated isolates revealed no close correlation between mutation types and levels of streptomycin resistance. No mutation was found in any of the susceptible isolates. The DHPLC results were completely consistent with those of sequencing. The DHPLC method devised in this study can be regarded as a useful and powerful tool for detection of streptomycin resistance. This is the first report to describe DHPLC analysis of mutations in the rpsL and rrs genes of M. tuberculosis in a large number of clinical isolates.
Microbes and Infection 01/2007; 9(14-15):1538-44. · 2.92 Impact Factor
[show abstract][hide abstract] ABSTRACT: To identify mycobacteria isolated from sputa of a 51-year-old female and a 72-year-old male patient with pneumoconiosis.
Mycobacteria species were isolated from sputa of a 51-year-old female. The culture was always negative in spite of positive smears before the final isolation in 1988. A 72-year-old male patient suffered from pneumoconiosis and the acid-fast bacillus was isolated by routine sputum examination in 2003. These two strains of acid-fast bacilli were identified as Mycobacterium heckeshornense by partial sequencing of 16S rRNA and rpoB and conventional methods (biochemical and routine culture methods).
These two strains grew on 1% Ogawa's slant medium at 37 degrees C and 42 degrees C, but not at 28 degrees C. They formed yellowish colonies in the dark (Scotochromogen). They were classified as a slowly growing Mycobacteria. As it was difficult to distinguish M. heckeshornense from M. xenopi by conventional methods including growth rate, temperature range of mycobacterial growth, light coloration reaction, biochemical and biological tests, virulence using guinea pigs and drug susceptibility test were further explored. Finally two were identified as M. heckeshornense by summing of these results.
Mycobacteria species that grow at 42 degrees C for four weeks, imply M. xenopi with a DDH method. It is essential to perform both sequencing of 16S rRNA and rpoB gene and a biochemical method for the purpose of distinguishing M. heckeshornense from M. xenopi.