Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Can Accurately Differentiate between Mycobacterium masilliense (M. abscessus subspecies bolletti) and M. abscessus ( Sensu Stricto )

Abstract

Among 36 Mycobacterium masilliense and 22 M. abscessus isolates identified by erm(41) PCR and sequencing analysis of rpoB and 23S rRNA genes, the rate of accurate differentiation between these two subspecies was 100% by cluster analysis of spectra generated by Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry. Mycobacterium abscessus complex, a rapidly growing mycobacterium, is the cause of an increasing number of community-and health care-associated infections in humans (1). The isolation of M. abscessus complex from patients with various clinical infections has been reported in many countries, including Taiwan (2-7). The M. abscessus complex comprises three closely related subspecies, namely, M. massiliense, M. bolletii, and M. abscessus (sensu stricto) (4,5,(7)(8)(9). Identification of M. abscessus complex members to the species level depends on sequencing analysis of several genes, including the erm(41) gene, the 23S rRNA gene, and several housekeeping genes (e.g., rpoB and hsp65) (7,(9)(10)(11)(12). A previous report found that erm(41) PCR can differentiate M. massiliense from M. abscessus and M. bolletii but that sequencing analysis of rpoB and hsp65 was less reliable at differentiating between the two (9). M. abscessus subsp. bolletti is now the recommended taxonomic name for M. massiliense (4, 12). Differences of in vitro susceptibilities to clarithromycin between M. massiliense (M. abscessus subsp. Bolletti) and M. abscessus (sensu stricto) isolates and of treatment response rates of lung diseases caused by these two species with clarithromycin-based antibiotic therapy have been reported (7-12). However, the use of molecular methods to differentiate among these subspecies is not possible in many routine microbiology laboratories.The use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is relatively new in the field of microbiology for species identification of yeasts and bacteria, including mycobacteria (13-17). Although MALDI-TOF MS has been shown to be a highly accurate method for identifying M. abscessus complex isolates to the species level, this method has not been shown to be able to differentiate between subspecies of the M. abscessus complex, i.e., M. massiliense (M. abscessus subsp. bolletti) and M. abscessus (sensu stricto) (15,16).In this study, we evaluated a total of 58 isolates of the M. abscessus complex obtained from various clinical specimens from patients treated during the period from January 2011 to December 2012 at the National Taiwan University Hospital, a 2,900-bed tertiary-care medical center in northern Taiwan. The isolates were presumptively identified as M. abscessus complex based on conventional biochemical methods as previously described (4-6).These isolates were further identified to the subspecies level by sequencing the erm(41) gene and by performing sequence analysis techniques targeting the rpoB and 23S rRNA genes. The following primer pairs were used: ermF (5=-GAC CGG GGC CTT CTT C...

Evaluation of Matrix-Assisted Laser Desorption Ionization−Time of Flight Mass Spectrometry for Identification of Mycobacterium abscessus Subspecies According to Whole-Genome Sequencing

“…(25,26). Peaks around 7,638 m/z or 7,668 m/z, described by Taiwanese and Japanese investigators, were not included in our SuperSpectra (26,27). This suggests that the two peaks in our data set might be ambiguous and not specific enough for identification, which might be due in part to the nature of the culture conditions used.…”

“…massiliense from the other two subspecies (23)(24)(25)(26)(27). However, discrepancies in the specific peak signals of the three subspecies were reported.…”

“…However, improvements in MALDI-TOF MS are needed to identify very close taxa that include the M. abscessus subspecies (23). A number of studies used spectrum analysis to show that several peaks differentiated M. abscessus at the subspe-cies level; these findings, however, were inconsistent (23)(24)(25)(26)(27). Possibly, these inconsistencies in subspecies profiles were determined by geographic location and related to the horizontal transfer of genes during the evolution of M. abscessus worldwide.…”

“…However, discrepancies in the specific peak signals of the three subspecies were reported. (25,26). Peaks around 7,638 m/z or 7,668 m/z, described by Taiwanese and Japanese investigators, were not included in our SuperSpectra (26,27).…”

MALDI-TOF MS for the Diagnosis of Infectious Diseases

“…MALDI-TOF MS is proving a useful tool for identification of mycobacteria with a few limitations, such as the inability to differentiate members of the Mycobacterium tuberculosis complex and some closely related species such as Mycobacterium chimaera and Mycobacterium intracellulare or Mycobacterium mucogenicum and Mycobacterium phocaicum. Although Mycobacterium abscessus and Mycobacterium massiliense may be challenging to differentiate, Teng et al did so using cluster analysis of spectra generated by the Biotyper system (30 ). MALDI-TOF MS is easier, less expensive, faster, and more accessible to routine clinical microbiology laboratories than traditional strategies for mycobacterial identification, which will likely make it the method of choice for their identification in the near future.…”