A nonphotochromogenic, rapidly growing Mycobacterium strain was isolated in pure culture from the sputum and the bronchoalveolar fluid of a patient with hemoptoic pneumonia by using axenic media and an amoebal coculture system. Both isolates grew in less than 7 days at 24 to 37°C with an optimal growth temperature of 30°C. The isolates exhibited biochemical and antimicrobial susceptibility profiles overlapping those of Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium immunogenum, indicating that they belonged to M. chelonae-M. abscessus group. They differed from M. abscessus in -galactosidase, -N-acetyl--glucosaminidase, and -glucuronidase activities and by the lack of nitrate reductase and indole production activities, as well as in their in vitro susceptibilities to minocycline and doxycycline. These isolates and M. abscessus differed from M. chelonae and M. immunogenum by exhibiting gelatinase and tryptophane desaminase activities. Their 16S rRNA genes had complete sequence identity with that of M. abscessus and >99.6% similarity with those of M. chelonae and M. immunogenum. Further molecular investigations showed that partial hsp65 and sodA gene sequences differed from that of M. abscessus by five and three positions over 441 bp, respectively. Partial rpoB and recA gene sequence analyses showed 96 and 98% similarities with M. abscessus, respectively. Similarly, 16S-23S rRNA internal transcribed spacer sequence of the isolates differed from that of M. abscessus by a A3G substitution at position 60 and a C insertion at position 102. Phenotypic and genotypic features of these two isolates indicated that they were representative of a new mycobacterial species within the M. chelonae-M. abscessus group. Phylogenetic analysis suggested that these isolates were perhaps recently derived from M. abscessus. We propose the name of "Mycobacterium massiliense" for this new species. The type strain has been deposited in the Collection Institut Pasteur as CIP 108297 T and in Culture Collection of the University of Göteborg, Göteborg, Sweden, as CCUG 48898 T .During the last few years, the number of nontuberculous mycobacteria (NTM) reported in various clinical situations has greatly increased because of opportunistic infections in immunocompromised patients and improved culture and identification techniques (18). The 16S rRNA gene sequence analysis of NTM led to the description of 40 new species since 1992 and contributed to the description of new clinical forms (19,35,43,44). Particularly, mycobacteria of the Mycobacterium chelonaeMycobacterium abscessus group (M. chelonae, M. abscessus, and Mycobacterium immunogenum) emerged as opportunistic pathogens (45, 47-49, 51), causing hypersensitivity pneumonitis in automobile production workers and chronic lung disease in elderly women with bronchiectasis and in young adults with cystic fibrosis (4,9,16,36,49). Precise species identification in this group of mycobacteria remains difficult. Only two biochemical tests, i.e., sodium chloride tolerance and utili...
Incidental blood agar-based recovery of Mycobacterium tuberculosis led us to further investigate this routine medium for primary isolation and culture of M. tuberculosis. Fifteen respiratory tract and eight lymph node Ziehl-Neelsen-positive specimens were inoculated in parallel into tubes containing egg-based medium and 5% sheep blood agar. Colonies appeared sooner on this medium than on the egg-based medium, but this difference was not significant (P ؍ 0.11, analysis of variance [ANOVA] test). Further experiments compared the growth of 38 respiratory and lymph node M. tuberculosis isolates when subcultured on the two media. After 6 days of incubation, 21 of 38 isolates had grown on blood agar, and the mean number of colonies was significantly greater on blood agar than on the egg-based medium (P < 0 0.001, ANOVA test). These results demonstrate that M. tuberculosis grows easily on blood agar within 1to 2 weeks, indicating that this basic medium is suitable for laboratory diagnosis of tuberculosis in addition to other media. Laboratories that routinely use prolonged incubations of blood plates, for example, for the recovery of Bartonella species, should consider the potential safety implications of encountering this highly infectious pathogen.Mycobacterium tuberculosis is a slow-growing bacterium that is the etiological agent of tuberculosis. Agar-based and eggbased media incorporating green malachite and Middlebrook broths or solid media are recommended as the "gold standard" for isolation, culture, and definite diagnosis of M. tuberculosis (6). Although there has been one anecdotal report of the isolation of M. tuberculosis on blood agar (M. Arvand, M. E. Mielke, T. Weinke, T. Regnath, and H. Hahn, Letter, Infection 26:254, 1998), microbiologists and medical students have been taught for decades that isolation of the organism requires a defined, egg-based medium, such as Lowenstein-Jensen medium (6).When trying to isolate Bartonella henselae from a lymph node by prolonged incubation on blood agar (5), we were surprised to isolate M. tuberculosis instead. The purpose of this preliminary work was to evaluate whether blood agar and prolonged incubation could support the growth of a variety of strains of M. tuberculosis obtained from different specimens. We now report that the current basic blood agar medium, used widely for nearly all primary isolations of bacteria, is at least as efficient as the widely recommended Lowenstein Jensen medium.Twenty three Ziehl-Neelsen-positive samples (15 respiratory tract specimens and 8 lymph node aspirates) were inoculated in parallel into tubes containing egg-based Coletsos medium, a local enriched formulation of Lowenstein-Jensen medium (bioMérieux, Marcy l'Etoile, France) and 5% sheep-blood agar (Bio Technologie Appliquée, Dinan, France). The respiratory tract specimens were digested using dithiothreitol and decontaminated using 2% NaOH before inoculation (6). Tubes were incubated at 37°C in a non-CO 2 atmosphere for 30 days, and they were examined every 2 days for the pre...
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