Mass spectrometry based methods for the discrimination and typing of mycobacteria E. Shitikov a,⇑ , E. Ilina a , L. Chernousova b , A. Borovskaya a , I. Rukin a , M. Afanas’ev a , T. Smirnova b , A. Vorobyeva b , E. Larionova b , S. Andreevskaya b , M. Kostrzewa c , V. Govorun a a Research Institute for Physical–Chemical Medicine, Moscow, Russia b Central TB Research Institute of RAMS, Moscow, Russia c Bruker Daltonik GmbH, Bremen, Germany article info Article history: Available online 30 December 2011 Keywords: MALDI Mycobacterium Tuberculosis Direct bacterial profiling Nontuberculous mycobacteria Spoligotyping abstract Identification and typing of mycobacteria is very important for epidemiology, susceptibility testing and diagnostic purposes. This paper describes the development and validation of the alternative methods for species identification and typing of mycobacteria based on a matrix-assisted laser desorption/ioniza- tion time-of-flight mass-spectrometry (MALDI-ToF MS). Altogether there were 383 clinical isolates analyzed which include 348 strains of Mycobacterium tuberculosis complex (MTBC) (342 strains of M. tuberculosis and 6 strains of M. bovis) and 35 strains of nontuberculous mycobacteria (NTM) repre- sented by 16 different species. Direct bacterial profiling (DBP) by means of MALDI-ToF MS was carried out. Cluster analysis of DBP mass spectra divided them into two large separate groups corresponding to MTBC and NTM, and also demonstrated the possibility of isolate identification at the species level. Spoligotyping protocol based on mass spectrometry was developed and validated, it matched completely to classical spoligotyping data. Our results suggest that MALDI-ToF MS has potential as a rapid and repro- ducible platform for the identification and typing of Mycobacterium species. Ó 2011 Elsevier B.V. All rights reserved. 1. Introduction The genus Mycobacterium consists of approximately 140 heter- ogeneous species of rapid- and slow-growing bacilli. The number of infections caused by mycobacteria has increased over the past few decades. The Mycobacterium tuberculosis complex (MTBC) has become a major cause of death in many developing countries and continues to be a public health problem globally. Nontubercu- lous mycobacteria (NTM) are responsible for mycobacteriosis and many opportunistic infections in both immunocompromised and immunocompetent individuals (Glassroth, 2008). Accurate and definitive differentiation of MTBC and NTM has become important due to the rise of infections and antimicrobial resistance in this genus. Further molecular typing of MTBC isolates has greatly facil- itated the understanding of epidemiology of these pathogens. Identification of mycobacteria at the species level is important for epidemiological, public health and therapeutic reasons. Tradi- tionally, mycobacterium is identified by its phenotypic traits, such as morphological features, growth rates and preferred growth tem- perature, pigmentation and biochemical profiles. These methods are well established and standardized. However, testing remains to be a laborious, difficult and time-consuming task, requiring as long as 12 weeks for positive identification of the organism, and its results are often not reported in a timely manner to guide clin- ical decisions. In the last decade advances in molecular methods have facilitated the cheap, rapid and reliable identification of many Mycobacterium species by molecular means (Cook et al., 2003; Devulder et al., 2005; Hance et al., 1989; Russo et al., 2006). How- ever, these methods are still labor-intensive, narrowly focused and may be applicable only for a limited number of species. If further characterization of samples is required additional methods have to be applied, which usually means another instrument base. The goal of this study was to demonstrate a complex solution based on a matrix-assisted laser desorption/ionization time-of- flight mass-spectrometry (MALDI-ToF MS) suitable for a rapid and accurate species identification and typing of mycobacteria. Recently, direct bacterial profiling (DBP) by means of MALDI- ToF MS has been suggested as an efficient approach for a rapid and accurate classification and species identification of microbes (Leuschner et al., 2004; Lynn et al., 1999; Mandrell et al., 2005; Mellmann et al., 2008) overcoming the existing limitations of clas- sical biochemical methods. This technique based on analysis of un- ique mass spectra produced by proteins extracted from microbial 1567-1348/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2011.12.013 ⇑ Corresponding author. Address: Research Institute for Physical–Chemical Medicine, 119435 Malaya Pirogovskaya str., 1a, Moscow, Russia. Tel.: +7 095246 45 70; fax: +7 095246 45 01. E-mail address: egorshtkv@gmail.com (E. Shitikov). Infection, Genetics and Evolution 12 (2012) 838–845 Contents lists available at SciVerse ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid