Ann Clin Microbiol.  2016 Dec;19(4):110-120. 10.5145/ACM.2016.19.4.110.

Taxonomic Identification of Bacillus Species Using Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry

• Yu WS
• Lee KM
• Hwang KJ

• KMID: 2362957
• DOI: http://doi.org/10.5145/ACM.2016.19.4.110

Abstract

BACKGROUND
In this study, we compared various methods of taxonomic identification of Bacillus strains: biochemical methods, 16S rRNA gene sequencing, and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). We also developed a pathogen- isolate resource database, thus increasing the identification rate when using MALDI-TOF MS.
METHODS
Thirty Bacillus strains were obtained from the NCCP (National Culture Collection for Pathogens) and were identified using the VITEK 2 system (bio-Mérieux, France), API kit (bioMérieux, France), 16S rRNA gene sequencing, and MALDI-TOF MS. The pathogenicity of Bacillus cereus was confirmed through the identification of virulent genes using a multiplex PCR, and both protein extraction for protein profiling in MALDI-TOF MS and repetitive-sequence fingerprinting were performed.
RESULTS
The identification rates at the species level were 40%, 80%, and 76.3% for the VITEK 2 system (bioMérieux), 16S rRNA gene sequencing, and MALDI-TOF MS, respectively. When the major spectrum-profiling dendrogram was compared with the phylogenetic tree, which was constructed based on the 16S rRNA gene sequences and rep-PCR fingerprinting, the classifications were confirmed to be effective.
CONCLUSION
Identification of Bacillus strains using MALDI-TOF MS was more effective than that using the VITEK 2 system (bioMérieux), but was similar to that using 16S rRNA gene sequencing. Continual addition to a proteome-based database can result in increased identification rates for MALDI-TOF MS.

Keyword

Bacillus speciess; Identification; MALDI-TOF MS

MeSH Terms

Bacillus cereus
Bacillus*
Classification
Dermatoglyphics
Genes, rRNA
Mass Spectrometry*
Multiplex Polymerase Chain Reaction
Trees
Virulence

Figure

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  Fig. 1. Representative spectra from Bacillus strains supplemented in the database by MALDI-TOF MS. The intensity is shown as a percentage of the total intensity on the y-axis, and the mass to charge ration (m/z) is shown on the x-axis. (A) Bacillus cereus NCCP10841 DSMZ32. (B) B. cereus NCCP14796 entFM, CER. (C) B. cereus NCCP15909 entFM, nheA, cytK. (D) B. thuringiensis NCCP11234.(E) B. subtillis NCCP10908. (F) B. licheniformis NCCP11231.

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  Fig. 2. Major spectrum profile (MSP) dendrogram of Bacillus strains supplemented in the database.

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  Fig. 3. Comparison of 16S rRNA gene, repetitive-sequence based PCR fingerprinting and MALDI-TOF MS analysis in Bacillus strains. Phylogenetic tree based by 16S rRNA gene with UPGMA method (A), rep-PCR fingerprinting (B) and MALDI-TOF MS (C).

Reference

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