Identification of Francisella tularensis by Whole-Cell Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry: Fast,Reliable, Robust,and Cost-Effective Differentiation on Species and Subspecies Levels

Francisella tularensis, the causative agent of tularemia, is a potential agent of bioterrorism. The phenotypic discrimination of closely related, but differently virulent, Francisella tularensis subspecies with phenotyping methods is difficult and time-consuming, often producing ambiguous results. As a fast and simple alternative, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was applied to 50 different strains of the genus Francisella to assess its ability to identify and discriminate between strains according to their designated species and subspecies. Reference spectra from five representative strains of Francisella philomiragia, Francisella tularensis subsp. tularensis, Francisella tularensis subsp. holarctica, Francisella tularensis subsp. mediasiatica, and Francisella tularensis subsp. novicida were established and evaluated for their capability to correctly identify Francisella species and subspecies by matching a collection of spectra from 45 blind-coded Francisella strains against a database containing the five reference spectra and 3,287 spectra from other microorganisms. As a reference method for identification of strains from the genus Francisella, 23S rRNA gene sequencing was used. All strains were correctly identified, with both methods showing perfect agreement at the species level as well as at the subspecies level. The identification of Francisella strains by MALDI-TOF MS and subsequent database matching was reproducible using biological replicates, different culture media, different cultivation times, different serial in vitro passages of the same strain, different preparation protocols, and different mass spectrometers.Francisella tularensis is the causative agent of the zoonotic disease tularemia. Beside its medical and veterinary impact, it possesses a high potential to be used for bioterrorist attacks (7). The World Health Organization (WHO) has modeled a bioterrorist attack with Francisella tularensis leading to very high costs for society ($5.4 billion if 100,000 individuals are affected) due to the extreme infectivity of the pathogen. Tularemia appears in various clinical forms depending on the virulence of the involved F. tularensis strain as well as the route and dose of inoculation.The genus Francisella was recently rearranged (12, 23) and is currently divided into four species, including F. tularensis and F. philomiragia. The species Francisella tularensis is further subdivided into four subspecies, F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, F. tularensis subsp. mediasiatica, and F. tularensis subsp. novicida, all four showing significant differences in virulence regarding animal and human infections (28). F. tularensis subsp. tularensis (type A), which is almost exclusively found in North America, and F. tularensis subsp. holarctica (type B), which is endemic in areas all over the northern hemisphere, represent the two most virulent and clinically relevant subspecies. Several strains of F. tularensis subsp. mediasiatica have been isolated in Central Asia, but little is known about their virulence in humans. F. tularensis subsp. novicida is rarely isolated from human specimens but can cause a tularemia-like disease in immunocompromised individuals (5).Differentiation of the highly virulent F. tularensis subsp. tularensis from the less virulent F. tularensis subsp. holarctica is of substantial clinical interest (30) but may be even more important regarding the potential use of F. tularensis as a biological warfare agent.Identification and differentiation of bacteria based on the polymorphism of the 16S and 23S rRNA genes have become widely accepted (18). Although 16S rRNA gene sequencing is accepted as the reference method for species identification (3, 6, 8), 23S rRNA gene sequencing seems to be more suitable for the identification of F. tularensis subspecies (W. Splettstoesser, E. Seibold, E. Zeman, K. H. Trebesius, and A. Podbielski, submitted for publication).Cultivation of F. tularensis is fastidious. It has high requirements for the growth media used for its cultivation (e.g., Thayer-Martin and cysteine heart agar CHA]), and cultivation may take up to 10 days (29). Identification and differentiation of Francisella strains by traditional phenotyping are difficult, often leading to ambiguous results. Additionally these methods include prolonged cultivation, thereby increasing the risk for laboratory-acquired tularemia (29).Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been successfully applied to identify bacterial species (2, 10, 13, 15, 20, 22, 24, 25) by measuring the mass of peptides and small proteins from whole cells. The resulting protein spectra are postulated to be characteristic for each bacterial species. The available literature regarding the identification of and discrimination between subspecies or even single strains by MALDI-TOF MS is limited (1, 24). Francisella tularensis subspecies were successfully discriminated with surface-enhanced laser desorption ionization-time of flight MS (SELDI-TOF MS) (19, 26), a modified MALDI-TOF MS method which allows selective absorption of proteins on a chromatographic array surface prior to MS (31, 32). Compared to SELDI-TOF MS, MALDI-TOF MS is less time-consuming and more cost-effective. Furthermore, there are several databases available for MALDI-TOF MS, which allow for matching spectra of unknown isolates with reference spectra.Although there are still concerns regarding the reproducibility of MALDI-TOF MS under different cultivation conditions, recent publications showed good reproducibility for various cultivation conditions in several bacterial groups (22, 24). A recent international study achieved 98.75% interlaboratory reproducibility regarding the identification of 60 blind-coded nonfermenting bacterial samples (21).The aim of our study was to identify and differentiate strains of the genus Francisella by the use of MALDI-TOF MS at the subspecies level by combining MALDI-TOF MS with dedicated bioinformatics and statistical methods, i.e., database search, pattern-matching algorithm, cluster analysis, and principal component analysis (PCA). Initial spectra from representative strains of each Francisella species and subspecies were used to set up database entries for reidentification of F. tularensis strains. This database was evaluated with 45 blind-coded Francisella strains that were also analyzed by 23S rRNA gene sequencing. Reproducibility of the method was tested with various growth media, cultivation times, numbers of strain passages, protein extraction protocols, and mass spectrometers.