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.