So far, genetic diversity among strains within Mycobacterium massiliense has rarely been studied. To investigate the genetic diversity among M. massiliense, we conducted phylogenetic analysis based on hsp65 (603-bp) and rpoB (711-bp) sequences from 65 M. massiliense Korean isolates. We found that hsp65 sequence analysis could clearly differentiate them into two distinct genotypes, Type I and Type II, which were isolated from 35 (53.8%) and 30 patients (46.2%), respectively. The rpoB sequence analysis revealed a total of four genotypes (R-I to R-IV) within M. massiliense strains, three of which (R-I, R-II and R-III) correlated with hsp65 Type I, and other (R-IV), which correlated with Type II. Interestingly, genotyping by the hsp65 method agreed well with colony morphology. Despite some exceptions, Type I and II correlated with smooth and rough colonies, respectively. Also, both types were completely different from one another in terms of MALDI-TOF mass spectrometry profiles of whole lipid. In addition, we developed PCR-restriction analysis (PRA) based on the Hinf I digestion of 644-bp hsp65 PCR amplicons, which enables the two genotypes within M. massiliense to be easily and reliably separated. In conclusion, two distinct hsp65 genotypes exist within M. massiliense strains, which differ from one another in terms of both morphology and lipid profile. Furthermore, our data indicates that Type II is a novel M. massiliense genotype being herein presented for the first time. The disparity in clinical traits between these two hsp65 genotypes needs to be exploited in the future study.

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Mycobacterium yongonense sp. nov., a slow-growing non-chromogenic species closely related to Mycobacterium intracellulare Among the slow-growing mycobacteria responsible for opportunistic infections, members of the Mycobacterium avium complex (MAC) are the non-tuberculous mycobacteria (NTM) most frequently isolated in clinical settings (Tortoli et al., 1999;Turenne et al., 2007; Yajko et al., 1995). Traditionally, the MAC included two species, Mycobacterium avium and Mycobacterium intracellulare As in other countries, members of the MAC are the most frequently isolated NTM in Korea. However, unique to Korea, M. intracellulare has been isolated more frequently than M. avium (Bai et al., 1993;Koh et al., 2005 Koh et al., , 2006Ryoo et al., 2008). We recently reported on diverse M. intracellulare-related strains showing genetic heterogeneity with one another in Korea, but distinct from those from other countries (Park et al., 2010).In the present study, we describe a novel clinical isolate that is closely related to M. intracellulare via a polyphasic taxonomic approach using a combination of biochemical tests and molecular analyses targeting the 16S rRNA Abbreviations: ITS1, internal transcribed spacer 1; MAC, Mycobacterium avium complex; MALDI-TOF MS, matrix-assisted laser desorptionionization time-of-flight mass spectrometry; NTM, non-tuberculous mycobacteria; PNB, p-nitrobenzoate; TCH, thiophene-2-carboxylic acid hydrazide.The GenBank/EMBL/DDBJ accession numbers for the partial 16S rRNA, hsp65, rpoB and ITS1 gene sequences of strain 05-1390 T are JF738056, JN605801, JF738053 and JF738054, respectively.A supplementary figure is available with the online version of this paper.

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A previously undescribed, slowly growing, scotochromogenic mycobacterium was isolated from a patient with symptomatic pulmonary infection during hsp65 sequence-based identification of Korean clinical isolates. Phenetic characteristics of this strain were generally similar to those of Mycobacterium nebraskense and Mycobacterium scrofulaceum. However, some phenetic characteristics differentiated it from these two species. Its 16S rRNA gene sequences were unique and phylogenetic analysis based on 16S rRNA gene sequences placed the organism in the slowly growing Mycobacterium group close to M. nebraskense and M. scrofulaceum. Its unique mycolic acid profiles and the results of phylogenetic analysis based on two independent alternative chronometer molecules, hsp65 and rpoB, confirmed the taxonomic status of this strain as representing a novel species. These data support the conclusion that this strain represents a novel mycobacterial species, for which the name Mycobacterium seoulense sp. nov. is proposed. The type strain is strain 03-19 T (=DSM 44998 T =KCTC 19146 T ).