Karen L. Wahl

Effect of Culture Conditions on Microorganism Identification by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

ABSTRACT Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has been used to identify bacteria based upon protein signatures. This research shows that while some different proteins are produced by vegetative bacteria when they are cultured in different growth media, positive identification with MALDI-TOF MS is still possible with the protocol established at the Pacific Northwest National Laboratory (K. H. Jarman, S. T. Cebula, A. J. Saenz, C. E. Petersen, N. B. Valentine, M. T. Kingsley, and K. L. Wahl, Anal. Chem. 72:1217-1223, 2000). A core set of small proteins remain constant under at least four different culture media conditions and blood agar plates, including minimal medium M9, rich media, tryptic soy broth (TSB) or Luria-Bertani (LB) broth, and blood agar plates, such that analysis of the intact cells by matrix-assisted laser desorption/ionization mass spectrometry allows for consistent identification.

Reproducibility of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry for replicate bacterial culture analysis

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to demonstrate the reproducibility of bacterial spectra collected on different days. The reproducibility of analysis by MALDI-MS of intact Escherichia coli and Bacillus atrophaeus is presented as a replicate culture study in which spectra were collected on ten different occasions over a three-month period and by two different operators. The analysis resulted in the detection of specific biomarkers in the m/z 2000-20 000 range. Some of the peaks in the Escherichia coli spectra are identified by comparison with other published work. All of the spectra obtained are reproducible over the course of the experiment, but operator variability does exist. The Escherichia coli spectra show operator variability while the Bacillus atrophaeus spectra do not. This work demonstrates the utility of MALDI in obtaining consistent spectra from bacteria over a period of time.

Extracting and visualizing matrix-assisted laser desorption/ionization time-of-flight mass spectral fingerprints.

We have developed a method for constructing and extracting matrix-assisted laser desorption/ionization (MALDI) fingerprints. This method is fully automated and statistically based, allowing a large number of spectra to be analyzed at a time in an objective manner. This method can be used to extract the fingerprint of a particular analyte from a spectrum containing multiple analytes. Therefore, this method lends itself well to real-world applications where samples to be analyzed are likely to be impure. We illustrate this method on experimental results from a series of studies of E. coli and B. atrophaeus MALDI time-of-flight mass spectrometry (TOFMS) fingerprints.

A new approach to automated peak detection

Abstract Spectral peak detection algorithms are often difficult to automate because they either rely on somewhat arbitrary rules, or are tuned to specific spectral peak properties. One popular approach detects peaks where signal intensities exceed some threshold. This threshold is typically set arbitrarily above the noise level or manually by the user. Intensity threshold-based methods can be sensitive to baseline variations and signal intensity. Another popular peak detection approach relies on matching the spectral intensities to a reference peak shape. This approach can be very sensitive to baseline changes and deviations from the reference peak shape. Such methods can be significantly challenged by modern analytical instrumentation where the baseline tends to drift, peaks of interest may have a low signal to noise (S/N) ratio, and no well-defined reference peak shape is available. We present a new approach for spectral peak detection that is designed to be generic and easily automated. Employing a histogram-based model for spectral intensity, peaks are detected by comparing the estimated variance of observations (the x -axis of the spectrum) to the expected variance when no peak is present inside some window of interest. We compare an implementation of this approach to two existing peak detection algorithms using a series of simulated spectra.

Use of an internal control for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of bacteria.

A method to aid in the analysis of bacterial samples of unknown concentration by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is demonstrated. It is shown that in MALDI analysis of bacteria, the intensities of resulting peaks in spectra are sensitive to the microbial concentration. At the high and low ends of the concentration range, no signal can be obtained, leaving very concentrated or very dilute samples indistinguishable. The addition of cytochrome c as an internal control allows the differentiation of these concentrated and dilute samples. The presence of the internal control causes only a 20% to 30% decrease in signal intensity when the bacterial concentration is optimum. However, the signal quality is improved when the internal control is added to some low concentrations of bacteria.

Mass spectrometry of low molecular mass solids by matrix-assisted laser desorption/ionization

Matrix-assisted laser desorption/ionization combined with time-of-flight mass spectrometry (MALDI/TOF-MS) was used for the analysis of low molecular mass compounds. Three classes of molecules were studied: organic acids, salts of oxyanions and amine-based chelating compounds. Mass spectra from samples of citric, propionic, butyric, oxalic and stearic acid; ethylenediaminetetraacetic acid (EDTA),N-(2-hydroxyethyl) ethylenediaminetriacetic acid (HEDTA), ethylenediamineN,N′-diacetic acid (EDDA) and nitrilotriacetic acid (NTA); and sulfate, nitrate, nitrite and phosphate salts were obtained. These species were analyzed alone and as mixtures in both the positive and negative ion modes. The organic acids and oxyanion salts displayed much stronger signals in the negative ion detection mode whereas chelating compounds, which contain basic amine functional groups, yielded stronger signals in the positive ion mode. This implies that detection sensitivity is often better for a particular ion mode in the analysis of small molecules containing limited classes of functional groups. In all analyses, the presence of high concentrations of sodium was found to quench the MALDI signals. To increase the detection sensitivity, some samples were processed through an ion-exchange column to remove sodium ions. This step was found to enhance the signal by two orders of magnitude over untreated samples.

Differentiation of Spores of Bacillus subtilis Grown in Different Media by Elemental Characterization Using Time-of-Flight Secondary Ion Mass Spectrometry

ABSTRACT We demonstrate the use of time-of-flight secondary ion mass spectrometry (TOF-SIMS) in a forensics application to distinguish Bacillus subtilis spores grown in various media based on the elemental signatures of the spores. Triplicate cultures grown in each of four different media were analyzed to obtain TOF-SIMS signatures comprised of 16 elemental intensities. Analysis of variance was unable to distinguish growth medium types based on 40Ca-normalized signatures of any single normalized element. Principal component analysis proved successful in separating the spores into groups consistent with the media in which they were prepared. Confusion matrices constructed using nearest-neighbor classification of the PCA scores confirmed the predictive utility of TOF-SIMS elemental signatures in identifying sporulation medium. Theoretical calculations based on the number and density of spores in an analysis area indicate an analytical sample size of about 1 ng, making this technique an attractive method for bioforensics applications.

Bayesian Integrated Microbial Forensics

ABSTRACT In the aftermath of the 2001 anthrax letters, researchers have been exploring ways to predict the production environment of unknown-source microorganisms. Culture medium, presence of agar, culturing temperature, and drying method are just some of the broad spectrum of characteristics an investigator might like to infer. The effects of many of these factors on microorganisms are not well understood, but the complex way in which microbes interact with their environments suggests that numerous analytical techniques measuring different properties will eventually be needed for complete characterization. In this work, we present a Bayesian statistical framework for integrating disparate analytical measurements. We illustrate its application to the problem of characterizing the culture medium of Bacillus spores using three different mass spectral techniques. The results of our study suggest that integrating data in this way significantly improves the accuracy and robustness of the analyses.

Evaluation of sampling tools for environmental sampling of bacterial endospores from porous and nonporous surfaces

Aims:  Having and executing a well‐defined and validated sampling protocol is critical following a purposeful release of a biological agent for response and recovery activities, for clinical and epidemiological analysis and for forensic purposes. The objective of this study was to address the need for validated sampling and analysis methods called out by the General Accounting Office and others to systematically compare the collection efficiency of various swabs and wipes for collection of bacterial endospores from five different surfaces, both porous and nonporous. This study was also designed to test the collection and extraction solutions used for endospore recovery from swabs and wipes.

Detection of agar, by analysis of sugar markers, associated with Bacillus anthracis spores, after culture.

Detection of small quantities of agar associated with spores of Bacillus anthracis could provide key information regarding its source or growth characteristics. Agar, widely used in growth of bacteria on solid surfaces, consists primarily of repeating polysaccharide units of 3,6-anhydro-l-galactose (AGal) and galactose (Gal) with sulfated and O-methylated galactoses present as minor constituents. Two variants of the alditol acetate procedure were evaluated for detection of potential agar markers associated with spores. The first method employed a reductive hydrolysis step, to stabilize labile anhydrogalactose, by converting to anhydrogalactitol. The second eliminated the reductive hydrolysis step simplifying the procedure. Anhydrogalactitol, derived from agar, was detected using both derivatization methods followed by gas chromatography-mass spectrometry (GC-MS) analysis. However, challenges with artifactual background (reductive hydrolysis) or marker destruction (hydrolysis) respectively lead to the use of an alternative agar marker. A minor agar component, 6-O-methyl galactose (6-O-M gal), was readily detected in agar-grown but not broth-grown bacteria. Detection was optimized by the use of gas chromatography-tandem mass spectrometry (GC-MS-MS). With appropriate choice of sugar marker and analytical procedure, detection of sugar markers for agar has considerable potential in microbial forensics.