title: Establishment and application of an analytical in-house database (IHDB) for rapid discrimination of Bacillus subtilis group (BSG) using whole-cell MALDI-TOF MS technology.
abstract: Members of the Bacillus subtilis group (BSG) possess industrial applicability; unfortunately, B. subtilis and its phylogenetically closest species are indistinguishable from one another using 16S rDNA sequencing, physiological and biochemical tests. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a relatively novel technique for the fast and reliable identification of microorganisms. The aim of this study was to construct a unique analytical in-house database (IHDB) for BSG discrimination based on whole-cell protein fingerprinting using MALDI-TOF MS, as well as to discover biomarkers from the MS peaks to generate a classification model for further differentiation using the ClinProTools software. Type strains of 12 species (included five subspecies) of the BSG were used to build a main spectrum profile (MSP) to create an IHDB under the optimized parameters. The BSG isolates obtained from partial recA gene sequencing were used for IHDB validation. A total of 84 (100%) isolates were correctly identified to the species level and had high score values (mean score: 2.52). However, the IHDB had ambiguous identification at the subspecies level of Bacillus amyloliquefaciens. After implementation of the classification models, the strains could be clearly differentiated. We have successfully developed a rapid, accurate and cost-effective platform for the species- and subspecies-level discrimination of BSG based on the implementation of the IHDB and coupled with ClinProTools, which can be employed as an alternative technology to DNA sequencing and applied for efficient quality control of the microbial agent.
authors: Bioresource Collection and Research Center, Food Industry Research and Development Institute, 331 Shih-Pin Road, Hsinchu, 30062, Taiwan.
authors: Chien-Hsun
authors: CH
authors: Huang
authors: Bioresource Collection and Research Center, Food Industry Research and Development Institute, 331 Shih-Pin Road, Hsinchu, 30062, Taiwan.
authors: Lina
authors: L
authors: Huang
authors: National Chiayi University, Department of Animal Science, 300 Syuefu Rd., Chiayi City, 60004, Taiwan.
authors: Mu-Tzu
authors: MT
authors: Chang
authors: National Chiayi University, Department of Animal Science, 300 Syuefu Rd., Chiayi City, 60004, Taiwan. Electronic address: ckl@mail.ncyu.edu.tw.
authors: Kuo-Lung
authors: KL
authors: Chen
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics / Q000302:isolation & purification*; D019992:Databases as Topic; D005798:Genes, Bacterial; D010802:Phylogeny; D011938:Rec A Recombinases / Q000235:genetics; D015203:Reproducibility of Results; D012984:Software; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013223:Statistics as Topic
chemical_list: D011938:Rec A Recombinases
keywords: Bacillus subtilis group; Biomarkers; MALDI-TOF MS; Rapid discrimination; Species and subspecies-level; recA sequencing