Nancy B. Valentine

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.

Kinetics of U(VI) reduction by a dissimilatory Fe(III)‐reducing bacterium under non‐growth conditions

Dissimilatory metal-reducing microorganisms may be useful in processes designed for selective removal of uranium from aqueous streams. These bacteria can use U(VI) as an electron acceptor and thereby reduce soluble U(VI) to insoluble U(IV). While significant research has been devoted to demonstrating and describing the mechanism of dissimilatory metal reduction, the reaction kinetics necessary to apply this for remediation processes have not been adequately defined. In this study, pure culture Shewanella alga strain BrY reduced U(VI) under non-growth conditions in the presence of excess lactate as the electron donor. Initial U(VI) concentrations ranged from 13 to 1680 microM. A maximum specific U(VI) reduction rate of 2.37 micromole-U(VI)/(mg-biomass h) and Monod half-saturation coefficient of 132 microM-U(VI) were calculated from measured U(VI) reduction rates. U(VI) reduction activity was sustained at 60% of this rate for at least 80 h. The initial presence of oxygen at a concentration equal to atmospheric saturation at 22 degrees C delays but does not prevent U(VI) reduction. The rate of U(VI) reduction by BrY is comparable or better than rates reported for other metal reducing species. BrY reduces U(VI) at a rate that is 30% of its Fe(III) reduction rate.

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.

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.

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.

Identification of bacterial spores using statistical analysis of Fourier transform infrared photoacoustic spectroscopy data.

Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) has been applied for the first time to the identification and speciation of bacterial spores. A total of forty specimens representing five strains of Bacillus spores (Bacillus subtilis ATCC 49760, Bacillus atrophaeus ATCC 49337, Bacillus subtilis 6051, Bacillus thuringiensis subsp. kurstaki, and Bacillus globigii Dugway) were analyzed. Spores were deposited, with minimal preparation, into the photoacoustic sample cup and their spectra recorded. Principal component analysis (PCA), classification and regression trees (CART), and Mahalanobis distance calculations were used on this spectral library to develop algorithms for step-wise classification at three levels: (1) bacterial/nonbacterial, (2) membership within the spore library, and (3) bacterial strain. Internal cross-validation studies on library spectra yielded classification success rates of 87% or better at each of these three levels. Analysis of fifteen blind samples, which included five samples of spores already in the spectral library, two samples of closely related Bacillus globigii 01 spores not in the library, and eight samples of nonbacterial materials, yielded 100% accuracy in distinguishing among bacterial/nonbacterial samples, membership in the library, and bacterial strains within the library.

Identification of Sporulated and Vegetative Bacteria Using Statistical Analysis of Fourier Transform Mid-infrared Transmission Data

A combined mid-infrared spectroscopic/statistical modeling approach for the discrimination and identification, at the strain level, of both sporulated and vegetative bacterial samples is presented. Transmission mode spectra of bacteria dried on ZnSe windows were collected using a Fourier transform mid-infrared (FT-IR) spectrometer. Five Bacillus bacterial strains (B. atrophaeus 49337, B. globigii Dugway, B. thuringiensis spp. kurstaki 35866, B. subtilis 49760, and B. subtilis 6051) were used to construct a reference spectral library and to parameterize a four-step statistical model for the systematic identification of bacteria. The statistical methods used in this initial feasibility study included principal component analysis (PCA), classification and regression trees (CART), andMahalanobis distance calculations. Internal cross-validation studies successfully classified 100% of the samples into their correct physiological state (sporulated or vegetative) and identified 67% of the samples correctly as to their bacterial strain. Analysis of thirteen blind samples, which included reference and other bacteria, nonbiological materials, and mixtures of both nonbiological and bacterial samples, yielded comparable accuracy. The primary advantage of this approach is the accurate identification of unknown bacteria, including spores, in a matter of minutes.

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.