Principal component analysis of MALDI TOF MS mass spectra separates M. abscessus (sensu stricto) from M. massiliense isolates.

BACKGROUND: The discrimination of the members of the Mycobacterium abscessus complex is of clinical interest because one of the subspecies, M. massiliense, exhibits higher rates of response to antibiotic treatment for lung infection than do the other members of that complex. M. abscessus complex contains three subspecies that are laborious to identify; therefore, a routine diagnostic tool would be worthwhile. RESULTS: We used principal component analysis, hierarchical cluster analysis, and single-peak analysis to examine peak lists derived from matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) mass spectra of 50 clinical M. abscessus complex isolates, including 28 M. abscessus (sensu stricto), 19 M. massiliense, and 3 M. bolletii isolates grown in mycobacterium growth indicator tube liquid medium and prepared with a bead-based protocol. Principal component analysis but not hierarchical cluster analysis separated M. abscessus (sensu stricto) isolates and M. massiliense isolates into two clusters. Furthermore, single-peak analysis displayed 4 discriminating peaks that separated M. abscessus (sensu stricto) from M. massiliense isolates. M. bolletii isolates did not exhibit specific peaks but resembled the M. abscessus (sensu stricto) peak profile and also grouped within this principal component analysis cluster. Principal component analysis of all peak lists with the exclusion of the four discriminating peaks again separated M. abscessus (sensu stricto) from M. massiliense isolates, thus relativizing the importance of these peaks for subspecies identification. CONCLUSIONS: Principal component analysis of peak lists derived from MALDI TOF mass spectra is a robust and convenient method of discriminating M. massiliense isolates from the other members of the M. abscessus complex.

Characterization of rough and smooth morphotypes of Mycobacterium abscessus isolates from clinical specimens.

Mycobacterium abscessus, which consists of the two subspecies M. abscessus subspecies abscessus and M. abscessus subspecies bolletii, can produce rough or smooth colony morphologies. Here we analyzed 50 M. abscessus isolates cultured from the respiratory specimens of 34 patients, 28 (82%) of whom had cystic fibrosis (CF), with respect to their colony morphologies and antibiotic susceptibilities. The overall proportions of occurrences of the two morphotypes were similar, with specimens from 50% of the patients showing a rough and 38% showing a smooth morphotype. A total of 12% of the specimens from the patients showed both morphotypes simultaneously. At the subspecies level, the proportions of rough and smooth morphotypes differed substantially; 88% of rough morphotypes belonged to M. abscessus subspecies abscessus, and 85% of smooth morphotypes belonged M. abscessus subspecies bolletii. Inducible clarithromycin resistance due to the Erm(41) methylase, as well as high-level resistance to clarithromycin due to mutations within the rrl gene, occurred independently of the morphotype. The MIC50s of amikacin and cefoxitin were identical for the two morphotypes, whereas the MIC50s of tigecycline were 0.25 µg/ml for the rough morphotype and 2.0 µg/ml for the smooth morphotype. Our results show that the smooth morphotype was more dominant in respiratory specimens from CF patients than previously thought. With respect to resistance, colony morphology did not affect the susceptibility of Mycobacterium abscessus to the first-line antibiotics clarithromycin, amikacin, and cefoxitin.

Mycobacterium tuberculosis Is a Natural Ornithine Aminotransferase (rocD) Mutant and Depends on Rv2323c for Growth on Arginine.

Mycobacterium tuberculosis (Mtb) possesses a genetic repertoire for metabolic pathways, which are specific and fit to its intracellular life style. Under in vitro conditions, Mtb is known to use arginine as a nitrogen source, but the metabolic pathways for arginine utilization have not been identified. Here we show that, in the presence of arginine, Mtb upregulates a gene cluster which includes an ornithine aminotransferase (rocD) and Rv2323c, a gene of unknown function. Isotopologue analysis by using 13C- or 15N-arginine revealed that in Mtb arginine is not only used as nitrogen source but also as carbon source for the formation of amino acids, in particular of proline. Surprisingly, rocD, which is widespread in other bacteria and is part of the classical arginase pathway turned out to be naturally deleted in Mtb, but not in non-tuberculous mycobacteria. Mtb lacking Rv2323c showed a growth defect on arginine, did not produce proline from arginine, and incorporated less nitrogen derived from arginine in its core nitrogen metabolism. We conclude that the highly induced pathway for arginine utilization in Mtb differs from that of other bacteria including non-tuberculous mycobacteria, probably reflecting a specific metabolic feature of intracellular Mtb.