Amoebal Coculture of “Mycobacterium massiliense” sp. nov. from the Sputum of a Patient with Hemoptoic Pneumonia

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Abstract
A nonphotochromogenic, rapidly growingMycobacteriumstrain 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 ofMycobacterium abscessus,Mycobacterium chelonae, andMycobacterium immunogenum, indicating that they belonged toM. chelonae-M. abscessusgroup. They differed fromM. abscessusin β-galactosidase, β-N-acetyl-β-glucosaminidase, and β-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 andM. abscessusdiffered fromM. chelonaeandM. immunogenumby exhibiting gelatinase and tryptophane desaminase activities. Their 16S rRNA genes had complete sequence identity with that ofM. abscessusand >99.6% similarity with those ofM. chelonaeandM. immunogenum. Further molecular investigations showed that partialhsp65andsodAgene sequences differed from that ofM. abscessusby five and three positions over 441 bp, respectively. PartialrpoBandrecAgene sequence analyses showed 96 and 98% similarities withM. abscessus, respectively. Similarly, 16S-23S rRNA internal transcribed spacer sequence of the isolates differed from that ofM. abscessusby a A→G 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 theM. chelonae-M. abscessusgroup. Phylogenetic analysis suggested that these isolates were perhaps recently derived fromM. 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 108297Tand in Culture Collection of the University of Göteborg, Göteborg, Sweden, as CCUG 48898T.

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