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

Abstract

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.

FIG. 1.

(a) Comparison of the mass spectra of the five Francisella reference strains in the range from 3,800 to 11,200 Da. Exemplary masses (in daltons) are depicted. a.u., arbitrary units. (b) Three-dimensional PCA plot of the technical replicates of the five Francisella strains. Every dot represents a single technical replicate. The spectra of a particular Francisella reference strain are color coded: F. tularensis subsp. tularensis in red, F. tularensis subsp. holarctica in green, F. tularensis subsp. mediasiatica in blue, F. tularensis subsp. novicida in yellow, and F. philomiragia in pink.

FIG. 2.

Cluster analysis of MALDI-TOF MS spectra of all Francisella strains. Distance is displayed in relative units.

FIG. 3.

Cluster analysis of MALDI-TOF MS spectra derived from biological replicates of five representative Francisella strains. Distance is displayed in relative units. R_1, R_2, and R_3 represent the biological replicates.