Determination of post-culture processing with carbohydrates by MALDI-MS and TMS derivatization GC-MS.

Biological materials generally require stabilization to retain activity or viability in a dry form. A number of industrial products, such as vaccines, probiotics and biopesticides have been produced as dry preparations. The same methods and materials used for stabilizing commercial microbial products may be applicable to preserving biothreat pathogens in a dry form. This is a likely step that may be encountered when looking at samples from terrorism attempts since only spores, such as those from Bacillus anthracis, are inherently stable when dried. The stabilizers for microbial preparations generally include one or more small carbohydrates. Different formulations have been reported for different industrial products and are often determined empirically. However sugar alcohols (mannitol and sorbitol) and disaccharides (lactose, sucrose and trehalose) are the common constituents of these formulations. We have developed an analytical method for sample preparation and detection of these simple carbohydrates using two complementary analytical tools, MALDI-MS and GC-MS. The native carbohydrates and other constituents of the formulation are detected by MALDI-MS as a screening tool. A longer and more detailed analysis is then used to specifically identify the carbohydrates by derivatization and GC-MS detection. Both techniques were tested against ten different types of stabilization recipes with Yersinia pestis cell mass cultured on different media types used as the biological component. A number of additional components were included in these formulations including proteins and peptides from serum or milk, polymers (e.g. poly vinyl pyrrolidone - PVP) and detergents (e.g. Tween). The combined method was characterized to determine several figures of merit. The accuracy of the method was 98% for MALDI-MS and 100% for GC-MS. The repeatability for detection of carbohydrates by MALDI-MS was determined to be 96%. The repeatability of compound identification by GC-MS was determined by monitoring variation in retention time, which is vital for identification of isomeric carbohydrates. The figures of merit illustrate an effective and accurate method for mono and disaccharide detection independent of formulation. This meets our primary goal for method development as small carbohydrates are among the most common stabilizers employed.

Human norovirus infection of caco-2 cells grown as a three-dimensional tissue structure.

Human norovirus (hNoV) infectivity was studied using a three-dimensional model of large intestinal epithelium. Large intestine Caco-2 cells were grown in rotating wall vessel bioreactors for 18-21 days at 37 degrees C and then transferred to 24-well tissue culture plates where they were infected with GI.1 and GII.4 human noroviruses collected from human challenge trials and various outbreak settings, respectively. Compared with uninfected cells, transmission micrographs of norovirus-infected cells displayed evidence of shortening or total loss of apical microvilli, and vacuolization. Quantitative reverse transcription real-time PCR (qRT-PCR) indicated an approximate 2-3 log10 increase in viral RNA copies for the infected cells. A passage experiment examined both the ability for continued viral RNA and viral antigen detection. In the passaged samples 1.01x10(6) copies ml(-1) were detected by qRT-PCR. Immune electron microscopy using primary antibody to hNoV GI.1 capsids in conjunction with 6 nm gold-labelled secondary antibodies was performed on crude cellular lysates. Localization of antibody was observed in infected but not for uninfected cells. Our present findings, coupled with earlier work with the three-dimensional small intestinal INT407 model, demonstrate the utility of 3-D cell culture methods to develop infectivity assays for enteric viruses that do not readily infect mammalian cell cultures.

The effect of growth medium on B. anthracis Sterne spore carbohydrate content.

The expressed characteristics of biothreat agents may be impacted by variations in the culture environment, including growth medium formulation. The carbohydrate composition of B. anthracis spores has been well studied, particularly for the exosporium, which is the outermost spore structure. The carbohydrate composition of the exosporium has been demonstrated to be distinct from the vegetative form containing unique monosaccharides. We have investigated the carbohydrate composition of B. anthracis Sterne spores produced using four different medium types formulated with different sources of medium components. The amount of rhamnose, 3-O-methyl rhamnose and galactosamine was found to vary significantly between spores cultured using different medium formulations. The relative abundance of these monosaccharides compared to other monosaccharides such as mannosamine was also found to vary with medium type. Specific medium components were also found to impact the carbohydrate profile. Xylose has not been previously described in B. anthracis spores but was detected at low levels in two media. This may represent residual material from the brewery yeast extract used to formulate these two media. These results illustrate the utility of this method to capture the impact of growth medium on carbohydrate variation in spores. Detecting carbohydrate profiles in B. anthracis evidentiary material may provide useful forensic information on the growth medium used for sporulation.

Preservation of viable Francisella tularensis for forensic analysis.

As a preservation solution, (1%) ammonium chloride may be preferred over other conventionally used storage solutions because of its compatibility with analytical techniques such as Mass Spectrometry. In this study, ammonium chloride performed as well or better than phosphate buffered saline with Tween or Butterfields/Tween for preserving Francisella tularensis subsp. novicida.

Residual agar determination in bacterial spores by electrospray ionization mass spectrometry.

Presented here is an analytical method to detect residual agar from a bacterial spore sample as an indication of culturing on an agar plate. This method is based on the resolubilization of agar polysaccharide from a bacterial spore sample, enzymatic digestion, followed by electrospray ionization tandem mass spectrometry (ESI-MS(n)) analysis for detection of a specific agar fragment ion. A range of Bacillus species and strains were selected to demonstrate the effectiveness of this approach. The characteristic agar fragment ion was detected in the spores grown on agar that were washed from 1 to 5 times, irradiated or nonirradiated, and not in the spores grown in broth. A sample containing approximately 10(8) spores is currently needed for confident detection of residual agar from culture on agar plates in the presence of bacterial spores with a limit of detection of approximately 1 ppm agar spiked into a broth-grown spore sample. The results of a proficiency test with 42 blinded samples are presented demonstrating the utility of this method with no false positives and only three false negatives for samples that were below the detection level of the method as documented.

Chemometric analysis of multiple species of Bacillus bacterial endospores using infrared spectroscopy: discrimination to the strain level.

Previous work using infrared spectroscopy has shown potential for rapid discrimination between bacteria in either their sporulated or vegetative states, as well as between bacteria and other common interferents. For species within one physiological state, however, distinction is far more challenging, and requires chemometrics. In the current study, we have narrowed the field of study by eliminating the confounding issues of vegetative cells as well as growth media and focused on using IR spectra to distinguish only between different species all in the sporulated state. Using principal component analysis (PCA) and a classification method based upon similarity measurements, we demonstrate a successful identification rate to the species level of 85% for Bacillus spores grown and sporulated in a glucose broth medium.

The infrared spectra of Bacillus bacteria part I: vegetative Bacillus versus sporulated cells and the contributions of phospholipids to vegetative infrared spectra.

This paper highlights the distinctions between the infrared (IR) absorption spectra of vegetative versus sporulated Bacillus bacteria. It is observed that there are unique signatures clearly associated with either the sporulated or vegetative state and that vegetative cells (or cell debris) can contribute to the spore spectra. A distinct feature at approximately 1739 cm(-1) appears to be unique to vegetative cell spectra and can also be used as an indicator of vegetative cells or cell debris in the spore spectra. The data indicate that the band arises from a lipid-soluble species such as an ester or phospholipid carbonyl bond and are consistent with it being either phosphatidyl glycerol (PG) or phosphatidylethanolamine (PE), two major classes of phospholipids found in vegetative cells of Bacillus species. A companion work discusses bands associated with the sporulated state.

The infrared spectra of Bacillus bacteria part II: sporulated Bacillus--the effect of vegetative cells and contributions of calcium dipicolinate trihydrate, CaDP.3H2O.

Our previous paper showed that certain infrared (IR) peaks, e.g., the peak at 1739 cm(-1), are due to varying (trace) amounts of vegetative cells amongst the Bacillus spores and that these and other vegetative bands are associated with lipid-soluble compounds, likely an ester or phospholipid. This work investigates the infrared spectra of eight different sporulated Bacillus bacteria. For the endospores it is observed that peaks at 1441, 1277, and 1015 cm(-1) along with a distinct quartet of peaks at 766, 725, 701, and 659 cm(-1) are clearly associated with calcium dipicolinate trihydrate, CaDP.3H2O. It is emphasized that the spore peaks, especially the quartet, arise from the calcium dipicolinate trihydrate and not from dipicolinic acid or other dipicolinate hydrate salts. The CaDP.3H2O infrared peaks and the effects of hydration are studied using quantum chemistry in the PQS software package. The quartet is associated with many modes including contributions from the Ca2+ counterion and hydration waters including Ca-O-H bends, H2O-Ca-O torsions, and O-C-O bends. The 1441 and 1015 cm(-1) modes are planar pyridine modes with the 1441 cm(-1) mode primarily a ring C-N stretch and the 1015 cm(-1) mode primarily a ring C-C stretch.

Characterization of microorganisms by MALDI mass spectrometry.

Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for characterization and analysis of microorganisms, specifically bacteria, is described here as a rapid screening tool. The objective of this technique is not comprehensive protein analysis of a microorganism but rather a rapid screening of the organism and the accessible protein pattern for characterization and distinction. This method is based on the ionization of the readily accessible and easily ionizable portion of the protein profile of an organism that is often characteristic of different bacterial species. The utility of this screening approach is yet to reach its full potential but could be applied to food safety, disease outbreak monitoring in hospitals, culture stock integrity and verification, microbial forensics, or homeland security applications.

1H, 13C, and 15N resonance assignments of murine amelogenin, an enamel biomineralization protein.

Amelogenin is the predominant matrix protein in developing dental enamel. Making extensive use of residue specific 15N-labeled amino acids samples, the majority of the main and side chain resonances for murine amelogenin were assigned in 2% aqueous acetic acid at pH 3.0.

Bayesian-integrated microbial forensics.

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.

Preparation and evaluation of spore-specific affinity-augmented bio-imprinted beads.

A novel, affinity-augmented, bacterial spore-imprinted, bead material was synthesized, based on a procedure developed for vegetative bacteria. The imprinted beads were intended as a front-end spore capture/concentration stage of an integrated biological detection system. Our approach involved embedding bead surfaces with Bacillus thuringiensis kurstaki (Bt) spores (as a surrogate for Bacillus anthracis) during synthesis. Subsequent steps involved lithographic deactivation using a perfluoroether; spore removal to create imprint sites; and coating imprints with the lectin, concanavalin A, to provide general affinity. The synthesis of the intended material with the desired imprints was verified by scanning electron and confocal laser-scanning microscopy. The material was evaluated using spore-binding assays with either Bt or Bacillus subtilis (Bs) spores. The binding assays indicated strong spore-binding capability and a robust imprinting effect that accounted for 25% additional binding over non-imprinted controls. The binding assay results also indicated that further refinement of the surface deactivation procedure would enhance the performance of the imprinted substrate.

Differentiation of spores of Bacillus subtilis grown in different media by elemental characterization using time-of-flight secondary ion mass spectrometry.

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.

Bacterial analysis by MALDI-TOF mass spectrometry: an inter-laboratory comparison.

Bacterial analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has been demonstrated in numerous laboratories, and a few attempts have been made to compare results from different laboratories on the same organism. It has been difficult to understand the causes behind the observed differences between laboratories when different instruments, matrices, solvents, etc. are used. In order to establish this technique as a useful tool for bacterial identification, additional efforts in standardizing the methods by which MALDI mass spectra are obtained and comparisons of spectra from different instruments with different operators are needed. Presented here is an extension of our previous single-laboratory reproducibility study with three different laboratories in a controlled experiment with aliquots of the same bacterial culture, matrix stock solution, and calibrant standards. Using automated spectral collection of whole-cell bacteria and automated data processing and analysis algorithms, fingerprints from three different laboratories were constructed and compared. Nine of the ions appeared reproducibly within all three laboratories, with additional unique ions observed within each of the laboratories. An initial evaluation of the ability to use a fingerprint generated within one laboratory for bacterial identification of a sample from another laboratory is presented, and strategies for improving identification rates between laboratories is discussed.

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), and Mahalanobis 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.

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.

Analysis of whole bacterial cells by flow field-flow fractionation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The purpose of this study is to develop a novel bacterial analysis method by coupling the flow field-flow fractionation (flow FFF) separation technique with detection by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The composition of carrier liquid used for flow FFF was selected based on retention of bacterial cells and compatibility with the MALDI process. The coupling of flow FFF and MALDI-TOF MS was demonstrated for P. putida and E. coli. Fractions of the whole cells were collected after separation by FFF and further analyzed by MALDI-MS. Each fraction, collected over different time intervals, corresponded to different sizes and possibly different growth stages of bacteria. The bacterial analysis by flow FFF/MALDI-TOF MS was completed within 1 h with only preliminary optimization of the process.

Direct surface analysis of fungal species by matrix-assisted laser desorption/ionization mass spectrometry.

In this study various methods of sample preparation and matrices were investigated to determine optimum collection and analysis criteria for fungal analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Intact spores and/or hyphae of Aspergillus niger, Rhizopus oryzae, Trichoderma reesei and Phanerochaete chrysosporium were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The fungal samples were applied to the MALDI sample target as untreated, sonicated, or acid/heat treated samples, or blotted directly from the fungal culture with double-stick tape. Ferulic acid or sinapinic acid matrix solution was layered over the dried samples and analyzed by MALDI-MS. Statistical analysis showed that simply using double-stick tape to collect and transfer to a MALDI sample plate typically worked as well as the other preparation methods, and required the least sample handling.

Analysis of microbial mixtures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Many different laboratories are currently developing mass-spectrometric techniques to analyze and identify microorganisms. However, minimal work has been done with mixtures of bacteria. To demonstrate that microbial mixtures could be analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), mixed bacterial cultures were analyzed in a double-blind fashion. Nine different bacterial species currently in our MALDI-MS fingerprint library were used to generate 50 different simulated mixed bacterial cultures similar to that done for an initial blind study previously reported (Jarman, K. H.; Cebula, S. T.; Saenz, A. J.; Petersen, C. E.; Valentine, N. B.; Kingsley, M. T.; Wahl, K. L. Anal. Chem. 2000, 72, 1217-1223). The samples were analyzed by MALDI-MS with automated data extraction and analysis algorithms developed in our laboratory. The components present in the sample were identified correctly to the species level in all but one of the samples. However, correctly eliminating closely related organisms was challenging for the current algorithms, especially in differentiating Serratia marcescens, Escherichia coli, and Yersinia enterocolitica, which have some similarities in their MALDI-MS fingerprints. Efforts to improve the specificity of the algorithms are in progress.