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.

Phenotypical and Mass Spectral Assessment Methods for Identification of Some Contagious Mastitis Pathogens

“…agalactiae and 72% of CNS were identified by API system. The results tabulated in the current study, supported by similar and widespread data for clinical human and veterinary microbiology (Ilina et al, 2009;Lartigue et al, 2009; Science Publications Marklein et al, 2009;Nagy et al, 2009;Barreiro et al, 2010;Dubois et al, 2010;Ilina et al, 2010;Seibold et al, 2010;Moon et al, 2013;Raemy et al, 2013), confirmed that MALDI-TOF MS contributes in the fast identification of bacteria (one day versus five to eight days for conventional identification) and consequently the chance of an earlier treatment of subclinical and clinical mastitis with suitable antimicrobial agents will be carried out. In addition, in the dairy industry, MALDITOF MS can provides a faster, cheaper and more reliable identification of microorganisms isolated from milk of animals witth clinical and subclinical mastitis.…”

Verification of a Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Method for Diagnostic Identification of High-Consequence Bacterial Pathogens

“…F. tularensis cultures (100%) were differentiated at the species level in the locally generated custom database, while the proprietary Bruker Biotyper database identified only 56% of the F. tularensis cultures at the species level. This result agrees with the result of Seibold et al (16), who identified cultures of Francisella spp. to the species level with a supplemented Biotyper database.…”

Identification of Highly Pathogenic Microorganisms by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: Results of an Interlaboratory Ring Trial

“…Therefore, accurate assays for microbial identification are important to ensure proper medical intervention, either in the case of a natural outbreak or an intentional release. Such assays must be able to identify unambiguously a broad panel of potential threat microorganisms in different background matrices that may or may not be contaminated with non-HPB bacteria (11).Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a rapid, accurate, sensitive, and cost-effective method that offers an adequate alternative to genome-based approaches and that has been widely used for identification and typing of microorganisms in a clinical routine setup (12-19), as well as for HPB (20)(21)(22)(23)(24)(25)(26)(27). This method does …”

“…Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a rapid, accurate, sensitive, and cost-effective method that offers an adequate alternative to genome-based approaches and that has been widely used for identification and typing of microorganisms in a clinical routine setup (12)(13)(14)(15)(16)(17)(18)(19), as well as for HPB (20)(21)(22)(23)(24)(25)(26)(27). This method does not depend on exclusive consumables and has revealed high levels of reproducibility in both intralaboratory and interlaboratory studies (28,29).…”