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Title: Rapid Bacterial Identification Using Fourier Transform Infrared Spectroscopy

Abstract

Recent studies at Pacific Northwest National Laboratory (PNNL) using infrared spectroscopy combined with statistical analysis have shown the ability to identify and discriminate vegetative bacteria, bacterial spores and background interferents from one another. Since the anthrax releases in 2001, rapid identification of unknown powders has become a necessity. Bacterial endospores are formed by some Bacillus species as a result of the vegetative bacteria undergoing environmental stress, e.g. a lack of nutrients. Endospores are formed as a survival mechanism and are extremely resistant to heat, cold, sunlight and some chemicals. They become airborne easily and are thus readily dispersed which was demonstrated in the Hart building. Fourier Transform Infrared (FTIR) spectroscopy is one of several rapid analytical methods used for bacterial endospore identification. The most common means of bacterial identification is culturing, but this is a time-consuming process, taking hours to days. It is difficult to rapidly identify potentially harmful bacterial agents in a highly reproducible way. Various analytical methods, including FTIR, Raman, photoacoustic FTIR and Matrix Assisted Laser Desorption/Ionization (MALDI) have been used to identify vegetative bacteria and bacterial endospores. Each has shown certain areas of promise, but each has shortcomings in terms of sensitivity, measurement time or portability. IRmore » spectroscopy has been successfully used to distinguish between the sporulated and vegetative state. [1,2] It has also shown its utility at distinguishing between the spores of different species. [2-4] There are several Bacillus species that occur commonly in nature, so it is important to be able to distinguish between the many different species versus those that present an imminent health threat. The spectra of the different sporulated species are all quite similar, though there are some subtle yet reproducible spectroscopic differences. Thus, a more robust and reliable method is needed for differentiation. Using chemometrics, a classification scheme was developed and performed on samples sporulated in glucose broth. PNNL has demonstrated that vegetative bacteria and endospores have unique infrared (IR) signatures that can be used to identify to the species-, and in some cases, even to the strain-level. We have shown that the IR spectra of spores of different species tend to be quite similar, yet the small but reproducible differences in the spectra allow for a certain degree of differentiation. Further studies have shown that the culture medium can also have an effect on the spectra. For the distinction between vegetative and endospores, we have consistently observed a series of four peaks at 766, 725, 702, and a fairly sharp peak (FWHM 7 cm-1) at 660 cm-1, present only in the endospore spectra.« less

Authors:
; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
917962
Report Number(s):
PNNL-SA-53047
TRN: US200817%%1049
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: SPIE Newsroom
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; BACILLUS; BACTERIA; BACTERIAL SPORES; CLASSIFICATION; GLUCOSE; LASERS; NUTRIENTS; SENSITIVITY; SPECTRA; SPECTROSCOPY; SPORES

Citation Formats

Valentine, Nancy B., Johnson, Timothy J., Su, Yin-Fong, and Forrester, Joel B. Rapid Bacterial Identification Using Fourier Transform Infrared Spectroscopy. United States: N. p., 2007. Web. doi:10.1117/2.1200701.0559.
Valentine, Nancy B., Johnson, Timothy J., Su, Yin-Fong, & Forrester, Joel B. Rapid Bacterial Identification Using Fourier Transform Infrared Spectroscopy. United States. doi:10.1117/2.1200701.0559.
Valentine, Nancy B., Johnson, Timothy J., Su, Yin-Fong, and Forrester, Joel B. Thu . "Rapid Bacterial Identification Using Fourier Transform Infrared Spectroscopy". United States. doi:10.1117/2.1200701.0559.
@article{osti_917962,
title = {Rapid Bacterial Identification Using Fourier Transform Infrared Spectroscopy},
author = {Valentine, Nancy B. and Johnson, Timothy J. and Su, Yin-Fong and Forrester, Joel B.},
abstractNote = {Recent studies at Pacific Northwest National Laboratory (PNNL) using infrared spectroscopy combined with statistical analysis have shown the ability to identify and discriminate vegetative bacteria, bacterial spores and background interferents from one another. Since the anthrax releases in 2001, rapid identification of unknown powders has become a necessity. Bacterial endospores are formed by some Bacillus species as a result of the vegetative bacteria undergoing environmental stress, e.g. a lack of nutrients. Endospores are formed as a survival mechanism and are extremely resistant to heat, cold, sunlight and some chemicals. They become airborne easily and are thus readily dispersed which was demonstrated in the Hart building. Fourier Transform Infrared (FTIR) spectroscopy is one of several rapid analytical methods used for bacterial endospore identification. The most common means of bacterial identification is culturing, but this is a time-consuming process, taking hours to days. It is difficult to rapidly identify potentially harmful bacterial agents in a highly reproducible way. Various analytical methods, including FTIR, Raman, photoacoustic FTIR and Matrix Assisted Laser Desorption/Ionization (MALDI) have been used to identify vegetative bacteria and bacterial endospores. Each has shown certain areas of promise, but each has shortcomings in terms of sensitivity, measurement time or portability. IR spectroscopy has been successfully used to distinguish between the sporulated and vegetative state. [1,2] It has also shown its utility at distinguishing between the spores of different species. [2-4] There are several Bacillus species that occur commonly in nature, so it is important to be able to distinguish between the many different species versus those that present an imminent health threat. The spectra of the different sporulated species are all quite similar, though there are some subtle yet reproducible spectroscopic differences. Thus, a more robust and reliable method is needed for differentiation. Using chemometrics, a classification scheme was developed and performed on samples sporulated in glucose broth. PNNL has demonstrated that vegetative bacteria and endospores have unique infrared (IR) signatures that can be used to identify to the species-, and in some cases, even to the strain-level. We have shown that the IR spectra of spores of different species tend to be quite similar, yet the small but reproducible differences in the spectra allow for a certain degree of differentiation. Further studies have shown that the culture medium can also have an effect on the spectra. For the distinction between vegetative and endospores, we have consistently observed a series of four peaks at 766, 725, 702, and a fairly sharp peak (FWHM 7 cm-1) at 660 cm-1, present only in the endospore spectra.},
doi = {10.1117/2.1200701.0559},
journal = {SPIE Newsroom},
number = ,
volume = ,
place = {United States},
year = {Thu Feb 01 00:00:00 EST 2007},
month = {Thu Feb 01 00:00:00 EST 2007}
}
  • Fourier Transform Infrared Photoacoustic Spectroscopy (FTIR-PAS) has been applied for the first time to the identification and speciation of bacterial spores. With minimal preparation the spores were deposited into the photoacoustic sample cup and their spectra recorded. A total of 40 different samples of 5 different strains of Bacillus spores were analyzed: Bacillus subtilis ATCC 49760, Bacillus atrophaeus ATCC 49337, Bacillus subtilis 6051, Bacillus thuringiensis ssp. kurstaki, and Bacillus globigii Dugway. The statistical methods used included principal-component analysis (PCA), classification and regression trees (CART), and Mahalanobis-distance calculations. Internal cross-validation studies successfully classify the spores according to their bacterial strain inmore » 38 of 40 cases (95%) and 36 of 40 (90%) in cross-validation. Analysis of fifteen blind samples, which included library and other spores, and nonbacterial materials, resulted in correct strain classification the blind samples that were members of the library and correct rejection of the nonbacterial samples.« less
  • A stopped-flow rapid-mixing device interfaced with a rapid-scan FT-IR spectrometer and a diode-array UV-visible spectrophotometer permits the observation of reaction transient intermediates over a broad range of wavelengths at minimal cost. The system has been evaluated for both spectral regions with the use of two different chemical reaction systems. The presence of a transient intermediate is clearly indicated in one case. Unexpected reactivity was observed in the other case. This approach will allow the study of chemical reactions even when no spectral changes occur in the UV-visible region. {copyright} {ital 1999} {ital Society for Applied Spectroscopy}
  • A significant challenge to realize the full potential of nanotechnology for therapeutic and diagnostic applications is to understand and evaluate how live-cells interact with an external stimulus, e.g., a nanosized particle (NSP), and the toxicity and broad risk associated with these stimuli. NSPs are increasingly used in medicine with largely undetermined hazards in complex cell dynamics and environments. It is difficult to capture the complexity and dynamics of these interactions by following an omics-based approach exclusively, which are expensive and time-consuming. Additionally, this approach needs destructive sampling methods. Live-cell attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectrometry is well suited tomore » provide noninvasive approach to provide rapid screening of cellular responses to potentially toxic NSPs or any stimuli. Herein we review the technical basis of the approach, the instrument configuration and interface with the biological media, and various effects that impact the data, data analysis, and toxicity. Our preliminary results on live-cell monitoring show promise for rapid screening the NSPs.« less
  • A new apparatus for the in situ characterization of the rapid expansion of supercritical solutions (RESS) by Fourier transform infrared spectroscopy is presented. The infrared characterization is complemented by particle sizing with a scanning mobility particle sizer, by three-wavelengths-extinction measurements, and by scanning electron microscopy. Several examples show that a wide range of information about particle properties can be obtained with this setup. One new aspect is the possibility to expand into the vacuum which also allows us to investigate the conditions in the collision-free region before the Mach disk. These investigations elucidate that in the free jet region themore » solvent CO{sub 2} condenses to particles with mean radii >50 nm for pre-expansion pressures between 100-400 bar and temperatures between 298-398 K.« less