Mycobacterium massiliense Type II genotype leads to higher level of colony forming units and TNF-α secretion from human monocytes than Type I genotype.

Recently, we introduced a novel Mycobacterium massiliense Type II genotype from Korean patients, in which all isolates showed only a rough (R) colony morphotype. In this study, we sought to compare clinical factors and virulence potentials of two genotypes of M. massiliense, Type I and Type II. Patients infected with Type II tend to be younger at infection than those infected with Type I (56.7 vs 62.3, p = 0.051). Type II was more significantly related to R colony type than Type I (34.1% vs 94.1%, p < 0.001). The Type II strain showed significantly more colony forming units (CFUs) and higher levels of TNF-α secretion in infection of human monocytes than the Type I strain. The challenge of extracted glycopeptidolipid (GPL) into human monocytes indicated that the loss of GPL from the cell wall of the Type II genotype led to a higher level of TNF-α secretion in a toll-like receptor 2(TLR2)-dependent manner. Taken together, our data suggest that the M. massiliense Type II genotype shows higher virulence than Type I, which may be due to the induction of TNF-α via the loss of GPL from the Type II cell wall.

Discovery of a novel hsp65 genotype within Mycobacterium massiliense associated with the rough colony morphology.

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.