Novel Tools for High-Throughput Differentiation of Bacillus strains using Infrared Microspectroscopy.
Creators:Grasso, Elizabeth M.
Advisor:Rodriguez-Saona, Luis E.
Contributors:Yousef, Ahmed E.
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Series/Report no.:Food Science and Technology. Graduate student poster competition, 2007
Bacillus species are widely distributed in soil, water, and air and some species possess environmental and industrial significance. The ubiquity of this spore-forming genus allows it to potentially threaten the safety of a wide range of foods. Bacillus spores are resistant to processing and sanitation procedures, making their control of considerable importance in the food industry to prevent product spoilage and foodborne diseases. The objective of this study was to develop a rapid method for the differentiation, identification, and classification of Bacillus species at the strain level using infrared microspectroscopy and multivariate pattern-recognition techniques. Vegetative cells (104 CFU/mL) of Bacillus spp., including Bacillus cereus (4), B. mycoides (3), and B. thuringienisis (3), were filtered onto hydrophobic-grid membranes (HGM). The HGM were placed on Tryptic Soy Agar (TSA) and incubated at 42oC for 18-20h. HGM were removed from the agar, dried, and biomass of individual strains were measured by Attenuated Total Reflectance (ATR) IR microspectroscopy. Soft Independent Modeling of Class Analogy (SIMCA) models, generated from transformed spectra in the 1100-900 cm-1 region, exhibited clusters that permitted accurate strain-level classification of all isolates with zero-misclassifications. Major discrimination was related to signal from lipids likely present in the cellular membrane. Interclass distances, a measure of the distance between clusters based on factor loadings, provided further information regarding separation of classes. In conclusion, a simple IR microspectroscopy technique combined with multivariate analysis could provide the food industry with a rapid and reagent-free screening procedure to complement elaborate molecular identification methods.
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