Rapid identification of mycobacterial whole cells in solid and liquid culture media by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Mycobacterial identification is based on several methods: conventional biochemical tests that require several weeks for accurate identification, and molecular tools that are now routinely used. However, these techniques are expensive and time-consuming. In this study, an alternative method was developed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). This approach allows a characteristic mass spectral fingerprint to be obtained from whole inactivated mycobacterial cells. We engineered a strategy based on specific profiles in order to identify the most clinically relevant species of mycobacteria. To validate the mycobacterial database, a total of 311 strains belonging to 31 distinct species and 4 species complexes grown in Löwenstein-Jensen (LJ) and liquid (mycobacterium growth indicator tube MGIT]) media were analyzed. No extraction step was required. Correct identifications were obtained for 97% of strains from LJ and 77% from MGIT media. No misidentification was noted. Our results, based on a very simple protocol, suggest that this system may represent a serious alternative for clinical laboratories to identify mycobacterial species.The genus Mycobacterium encompasses over 100 species. The most important mycobacterial diseases are predominantly caused by the Mycobacterium tuberculosis complex. The incidence of other mycobacterial diseases due to nontuberculous mycobacteria (NTM) appears to be increasing in recent years due to the increasing number of immunocompromised individuals (1). Because the treatment of these infections differs depending on the isolated species, the correct and rapid identification of causative organisms is essential.Conventional methods for the identification of mycobacteria were classically based on biochemical tests. They required several weeks for adequate growth, and sometimes accurate identification was not possible. Difficulties, such as the lack of adequate reproducibility, the variability of phenotypes, and the fact that phenotype information is limited to common species, may lead to ambiguous or erroneous results (29). New strategies have been developed in the last decades using molecular biology tools (6, 10, 16, 24). The techniques based on DNA hybridization are sensitive, fast, and simple, but the available commercial assays (AccuProbe; Gen-Probe, San Diego, CA) are able to identify only four species and two complexes of mycobacteria (10). Techniques requiring amplification followed by a hybridization step on a solid support are more complete than probes, but commercially available kits are limited to 5 (GenoType MTBC; Hain Lifescience GmbH, Nehren, Germany), 16 (Inno-LiPa Mycobacteria v2; Innogenetics, Gent, Belgium), or 30 (GenoType Mycobacterium; Hain Lifescience GmbH, Germany) species (19, 22, 30). Systems based on sequencing or enzymatic restriction targeting the hsp65, 16S rRNA, sod, and rpoB genes allow good identification of all mycobacteria at the species level but remain limited to specialized laboratories (14, 17, 23, 31, 36, 37). In addition, they are expensive and time-consuming and require qualified operators (20). Recently, alternatives based on the analysis of mycolic acid by high-performance liquid chromatography (HPLC) (2) or electrospray ionization-tandem mass spectrometry analysis (28) have been proposed. However, these methods are still labor-intensive.Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) allows rapid identification of the most frequently isolated bacteria grown on solid medium by the identification of species-specific profiles obtained from isolated colonies (3-5). This technique is now routinely used in a few laboratories (26, 34). Some authors have used MALDI-TOF MS for rapid identification of Mycobacterium species (12, 18, 21). However, the techniques used require several steps, such as 16S rRNA gene-based techniques (18), cell extractions (12), or a statistical analysis (12, 21). In addition, the number of tested strains in these studies is less than 40, encompassing a maximum of 13 species. It was first reported by Hettick et al. that the analysis of mycobacterial whole cells by MALDI-TOF MS could be used for identification. However, the limited number of strains prevented the engineering of a useful database (11).Recently, we engineered a strategy to identify bacteria using MALDI-TOF MS, based on the choice of a limited number of species-specific profiles (3, 5). The aim of the present work is to extend this strategy to the identification of mycobacterial strains without cell extraction. This method will allow us to have a rapid, accurate, and inexpensive identification tool in routine laboratories. The first step was to build a complete database for mycobacterial species isolated in human pathology. This database was then validated by using clinical strains cultivated in solid and liquid media.