Direct identification of clinically relevant bacterial and yeast microcolonies and macrocolonies on solid culture media by Raman spectroscopy

Abstract

Abstract. Decreasing turnaround time is a paramount objective in clinical diagnosis. We evaluated the discrimination power of Raman spectroscopy when analyzing colonies from 80 strains belonging to nine bacterial and one yeast species directly on solid culture medium after 24-h (macrocolonies) and 6-h (microcolonies) incubation. This approach, that minimizes sample preparation and culture time, would allow resuming culture after identification to perform downstream antibiotic susceptibility testing. Correct identification rates measured for macrocolonies and microcolonies reached 94.1% and 91.5%, respectively, in a leave-one-strain-out cross-validation mode without any correction for possible medium interference. Large spectral differences were observed between macrocolonies and microcolonies, that were attributed to true biological differences. Our results, conducted on a very diversified panel of species and strains, were obtained by using simple and robust sample preparation and preprocessing procedures, while still confirming published results obtained by using more complex elaborated protocols. Instrumentation is simplified by the use of 532-nm laser excitation yielding a Raman signal in the visible range. It is, to our knowledge, the first side-by-side full classification study of microorganisms in the exponential and stationary phases confirming the excellent performance of Raman spectroscopy for early species-level identification of microorganisms directly from an agar culture. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

The use of microfluidics and dielectrophoresis for separation, concentration, and identification of bacteria

“…The classifications documented in Table 4 [30,32,33,35,36,38,100,118,119] while the majority of protein peaks increase or remain unchanged [36]. These results are supported by the work of Talukder et al [121] who used a quantitative Western immunoblot to determine concentrations of DNA and proteins of E. coli cells through the log and stationary phases.…”

“…Samples were grown in Luria-Bertani broth and growth phase was identified by optical density (OD) measurements. The majority of Raman peaks associated with RNA and DNA decreased as bacteria aged from the log to the stationary phase, which has been seen by many groups [30][31][32][33]35,38,118,119]. Moritz et al [36] also found that protein-related peaks increase or stay the same over the same timeframe.…”

“…In contrast, culture conditions of the human body vary from person to person. Researchers are aware of the discrepancy and have investigated the changes in Raman spectra due to factors such as temperature [29,30], growth phase [29][30][31][32][33][34][35][36][37][38], and growth media [29,30,33,38]. For the sake of this review, emphasis will be placed on the influence of growth media and phase on Raman spectra.…”

“…The influence of growth time or growth phase (metabolic state) on Raman spectra of bacteria has been studied by a number of researchers [29][30][31][32][33][34][35][36][37][38] Other articles have collected and compared Raman spectra of bacteria from different growth times, but have not specified growth stage. Choo-Smith et al [34] collected and compared Raman spectra of bacterial colonies grown on agar plates for 6, 12, and 24 hours at various colony depths using a confocal Raman microscope.…”

“…The influence of growth phase on Raman spectra has also been researched and includes direct spectral observation as well as statistical discriminatory methods [29][30][31][32][33][34][35][36][37][38]118,119]. Cell phases include lag, log, transition, stationary, and death phase.…”