Identification of Mycobacteria in Solid-Culture Media by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Abstract

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been introduced into the clinical microbiology laboratory as a rapid and accurate method to identify bacteria and yeasts. In this paper we describe our work on the use of MALDI-TOF MS for the identification of mycobacterial isolates. We developed a protocol for protein extraction from mycobacteria and utilized it to construct a database containing 42 clinically relevant type and reference strains of mycobacteria. The database was used to identify 104 clinical isolates of mycobacteria. All members of the Mycobacterium tuberculosis complex were identified accurately at the complex level but could not be separated at the species level. All other organisms were identified at the species level, with the exception of one strain of M. kansasii (accurately identified but with a low spectral score) and three pairs of closely related strains: M. abscessus and M. massiliense, M. mucogenicum and M. phocaicum, and M. chimaera and M. intracellulare. These pairs of organisms can currently be identified only by multilocus gene sequence analysis. We conclude that MALDI-TOF MS analysis can be incorporated into the work flow of the microbiology laboratory for rapid and accurate identification of most strains of mycobacteria isolated from solid growth media.The genus Mycobacterium consists of nearly 150 species, many of which are clinically significant. These organisms cause significant morbidity in humans, including pulmonary infections, skin and soft tissue infections, and disseminated disease. Rapid and accurate diagnosis of mycobacterial infections is of utmost importance due to the fact that inappropriate treatment may lead to drug resistance or unnecessary exposure to drug toxicities. Accurate identification in the microbiology laboratory is challenging because biochemical testing is slow and unable to identify less common species, molecular probes are available for a limited number of species, and gene sequencing is technically cumbersome and relatively expensive.Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is recognized as a powerful tool for the identification of bacteria and yeasts in the clinical laboratory (2,16,17,18,21). This technique allows identification of organisms on the basis of unique spectral fingerprints produced by extracted proteins (9). The method is relatively simple, rapid, and associated with significantly lower consumable costs than traditional microbiological identification methods (16). Although the MALDI-TOF MicroFlex LT mass spectrometer (Bruker Daltonics Inc., Billerica, MA) and associated software are expensive initially (approximately $200,000), the continuing consumable costs are inexpensive (less than $1 per isolate).There has been limited work thus far on the use of MALDI-TOF MS for the identification of mycobacteria (5, 6, 10). This paper describes our efforts to develop and validate MALDI-TOF MS for the identification of mycobacterial organ...

Rapid method for Mycobacterium tuberculosis identification using electrospray ionization tandem mass spectrometry analysis of mycolic acids

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Comparing inactivation protocols of Yersinia organisms for identification with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

“…[13] In our current study, we observed that ethanol inactivation yielded MALDI-TOF spectra of significantly higher quality than spectra derived from TFA inactivation, resulting in better [3,4,9] Brucella melitensis g-irradiation [7] Burkholderia spp. 80% TFA, 30 min [9] Francisella tularensis g-irradiation [7] Mycobacterium tuberculosis 95 C, 30-60 min [13,14] Yersinia pestis 70% ethanol, 60 min [10] identification scoring. These data may be linked to the better wetting properties across spots of 70% ethanol relative to 80% TFA.…”

Transplantation of human skin microbiota in models of atopic dermatitis

“…Bacterial protein extraction for MALDI-TOF mass spectrometry using the BioTyper (v3.1, Bruker Daltonics Inc.) was performed by the NIH Clinical Center microbiology lab using previously described methods (46), instrument settings, and calibration (47,48). BioTyper identification was supplemented by additional mass spectra profiles provided by several NIH-developed databases (46,49,50). Ten of the healthy control R. mucosa strains reported in Figure 1 were identified based solely on the unique colony morphology, UV light reactivity, and Gram stain characteristics that had been observed in the other R. mucosa isolates identified by MALDI-TOF analysis.…”