Proof of Principle for a Real-Time Pathogen Isolation Media Diagnostic: The Use of Laser-Induced Breakdown Spectroscopy to Discriminate Bacterial Pathogens and Antimicrobial-Resistant Staphylococcus aureus Strains Grown on Blood Agar
Laser-Induced Breakdown Spectroscopy (LIBS) is a rapid, in situ, diagnostic technique in which light emissions from a laser plasma formed on the sample are used for analysis allowing automated analysis results to be available in seconds to minutes. This speed of analysis coupled with little or no sample preparation makes LIBS an attractive detection tool. In this study, it is demonstrated that LIBS can be utilized to discriminate both the bacterial species and strains of bacterial colonies grown on blood agar. A discrimination algorithm was created based on multivariate regression analysis of spectral data. The algorithm was deployed on a simulated LIBS instrument system to demonstrate discrimination capability using 6 species. Genetically altered Staphylococcus aureus strains grown on BA, including isogenic sets that differed only by the acquisition of mutations that increase fusidic acid or vancomycin resistance, were also discriminated. The algorithm successfully identified all thirteen cultures used in this study in a time period of 2 minutes. This work provides proof of principle for a LIBS instrumentation system that could be developed for the rapid discrimination of bacterial species and strains demonstrating relatively minor genomic alterations using data collected directly from pathogen isolation media. 1. Introduction The goal of this work is to evaluate Laser-Induced Breakdown Spectroscopy (LIBS) as a tool for the rapid discrimination of bacterial cultures. LIBS is of interest for this application because of its speed of analysis, and because standard identification practices cannot easily distinguish all bacterial pathogen colonies. In LIBS, a laser pulse is focused onto a sample to vaporize and excite μg to ng amounts of material and generate a microplasma or laser spark. Light from the spark is collected and directed to a spectrometer to produce a spectrum that is recorded. The spectrum represents a combination of spectral signals from atoms and molecules of the samples and the surrounding atmosphere. Because the microplasma is formed by focused light, typically little to no sample preparation is required and, with automated analysis, results are available within seconds to minutes. LIBS is an analysis technique that is an outgrowth of atomic emission spectroscopy circa 1860 in which samples were placed in a flame and the colors observed were used for analysis [1]. Since these early experiments, plasma excitation sources such as the electrode spark and inductively coupled plasma have been developed. The first report of the use of a laser
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