Identification of Bacillus Spores by Matrix-Assisted Laser Desorption Ionization–Mass Spectrometry

Unique patterns of biomarkers were reproducibly characterized by matrix-assisted laser desorption ionization (MALDI)–mass spectrometry and were used to distinguish Bacillus species members from one another. Discrimination at the strain level was demonstrated for Bacillus cereus spores. Lipophilic biomarkers were invariant in Bacillus globigii spores produced in three different media and in B. globigii spores stored for more than 30 years. The sensitivity was less than 5,000 cells deposited for analysis. Protein biomarkers were also characterized by MALDI analysis by using spores treated briefly with corona plasma discharge. Protein biomarkers were readily desorbed following this treatment. The effect of corona plasma discharge on the spores was examined.     

Detection of Bacteriocins by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the detection of bacteriocins was investigated. A 30-s water wash of the sample on the MALDI-TOF MS probe was effective in removing contaminants of the analyte. This method was used for rapid detection of nisin, pediocin, brochocin A and B, and enterocin A and B from culture supernatants and for detection of enterocin B throughout its purification.     

Direct Identification of Bacteria in Positive Blood Culture Bottles by Matrix-Assisted Laser Desorption Ionisation Time-of-Flight Mass Spectrometry

Background

With long delays observed between sampling and availability of results, the usefulness of blood cultures in the context of emergency infectious diseases has recently been questioned. Among methods that allow quicker bacterial identification from growing colonies, matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry was demonstrated to accurately identify bacteria routinely isolated in a clinical biology laboratory. In order to speed up the identification process, in the present work we attempted bacterial identification directly from blood culture bottles detected positive by the automate.

Methodology/Principal Findings

We prospectively analysed routine MALDI-TOF identification of bacteria detected in blood culture by two different protocols involving successive centrifugations and then lysis by trifluoroacetic acid or formic acid. Of the 562 blood culture broths detected as positive by the automate and containing one bacterial species, 370 (66%) were correctly identified. Changing the protocol from trifluoroacetic acid to formic acid improved identification of Staphylococci, and overall correct identification increased from 59% to 76%. Lack of identification was observed mostly with viridans streptococci, and only one false positive was observed. In the 22 positive blood culture broths that contained two or more different species, only one of the species was identified in 18 samples, no species were identified in two samples and false species identifications were obtained in two cases. The positive predictive value of bacterial identification using this procedure was 99.2%.

Conclusions/Significance

MALDI-TOF MS is an efficient method for direct routine identification of bacterial isolates in blood culture, with the exception of polymicrobial samples and viridans streptococci. It may replace routine identification performed on colonies, provided improvement for the specificity of blood culture broths growing viridans streptococci is obtained in the near future.     

Characterization of Proteins Utilized in the Desulfurization of Petroleum Products by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF/MS) with delayed extraction is utilized in linear, reflected-ion and post-source decay (PSD) modes to directly characterize enzymes being developed for use in a petroleum desulfurization process. The DNA sequence for the genes isolated fromRhodococcussp. strain IGTS8 that produce three of the four enzymes under study had been previously reported with a discrepancy in residue assignments for one of the enzymes, dsz-C. The use of proteolytic digests followed by MALDI/TOF/MS with delayed extraction in the reflected-ion mode provided sequence-specific information with mass accuracies exceeding 40 ppm over a range of masses and signal-to-noise values. Peptide mapping of >80% of the residues was accomplished for all four proteins. The use of PSD established the true sequence for dsz-C, resolving the discrepancy in the literature. A posttranslational loss of N-terminal methionine was observed for each of the four proteins in linear MALDI/MS and was reconfirmed by peptide mapping for three of the proteins.     

Universal Sample Preparation Method for Characterization of Bacteria by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Mass spectrometry has been a very useful method to rapidly identify microorganisms associated with infectious diseases, detect bioterrorism threats, and discriminate among different subtypes of a pathogen. In this study, we developed a universal method for bacterial identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The effects on the mass spectrum of different experimental conditions, including the amount of bacterial cells used and treatment procedures with different solutions, matrix species, and solvents, were examined, and an optimized protocol was developed. Several different bacterial species, including Yersinia pestis, Escherichia coli, Burkholderia cepacia, Bacillus anthracis, and Staphylococcus aureus, which covered the gram-negative and -positive species and spore-producing and non-spore-producing species, were analyzed to evaluate the utility of the protocol. The results showed that five different species and different strains of the same species (9 strains of S. aureus and 10 strains of E. coli) could be discriminated clearly by their peak profiles in a mass range of 1,000 to 20,000 Da. This protocol is simple, rapid, and easy to perform; has excellent reproducibility; and is suitable for the construction of a mass spectrum fingerprinting database, which helps in fast bacterial identification via database searching.     

应用基质辅助激光电离飞行时间质谱快速鉴定临床酵母菌

目的研究基质辅助激光解析电离飞行时间质谱(Matrix-Assisted Laser Desorption Ionization-Time of Flight MassSpectrometry,MALDI-TOF-MS)用于快速检测鉴定临床分离的酵母菌的可行性。方法应用Bruker MALDI-TOF-MS和VITEK 2-compact系统分别鉴定150株临床分离的酵母菌,结果不一致的菌株通过基因序列测定来鉴定。结果 MALDI-TOF-MS快速准确鉴定出了150株临床酵母菌,鉴定符合率在属水平上为100%,种水平上为94%。结论基于MALDI-TOF-MS鉴定方法具有很好的可重复性和准确性,并且其检测成本较低,实验准备时间很短,MALDI-TOF-MS可以用于临床分离的酵母菌的快速鉴定。     

Rapid Identification of Vibrio parahaemolyticus by Whole-Cell Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Vibrio parahaemolyticus is a pathogenic marine bacterium that is the main causative agent of bacterial seafood-borne gastroenteritis in the United States. An increase in the frequency of V. parahaemolyticus-related infections during the last decade has been attributed to the emergence of an O3:K6 pandemic clone in 1995. The diversity of the O3:K6 pandemic clone and its serovariants has been examined using multiple molecular techniques including multilocus sequence analysis, pulsed-field gel electrophoresis, and group-specific PCR analysis. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has become a powerful tool for rapidly distinguishing between related bacterial species. In the current study, we demonstrate the development of a whole-cell MALDI-TOF MS method for the distinction of V. parahaemolyticus from other Vibrio spp. We identified 30 peaks that were present only in the spectra of the V. parahaemolyticus strains examined in this study that may be developed as MALDI-TOF MS biomarkers for identification of V. parahaemolyticus. We detected variation in the MALDI-TOF spectra of V. parahaemolyticus strains isolated from different geographical locations and at different times. The MALDI-TOF MS spectra of the V. parahaemolyticus strains examined were distinct from those of the other Vibrio species examined including the closely related V. alginolyticus, V. harveyi, and V. campbellii. The results of this study demonstrate the first use of whole-cell MALDI-TOF MS analysis for the rapid identification of V. parahaemolyticus.Recent food-borne illness outbreaks have emphasized the need for rapid, robust, and low-cost methods for microbial identification. Vibrio parahaemolyticus is one of several Vibrio species that cause human infection and occur in coastal estuarine and marine environments worldwide. V. parahaemolyticus causes gastroenteritis, wound infections, and septicemia upon exposure to contaminated water or contaminated undercooked seafood. In the United States, V. parahaemolyticus is the leading causative agent of bacterial seafood-borne gastroenteritis (8). Gastroenteritis-associated V. parahaemolyticus strains typically possess one or both of the thermostable direct hemolysin genes (tdh and trh); however, recent studies have indicated the presence of additional virulence-associated genes including two type III secretion systems (6, 7, 26, 28, 33). Following the emergence of the V. parahaemolyticus O3:K6 pandemic clone in 1995, there has been a rise in the number of reported V. parahaemolyticus-associated infections each year, making this species a pathogen of increasing concern (8, 11). The V. parahaemolyticus pandemic clone was first isolated from outbreaks in Asia in 1995 with the O3:K6 serotype and has since emerged with additional serotypes (30). The worldwide spread of the V. parahaemolyticus O3:K6 clone is a recognized international public health issue that requires the use of standardized methods for global monitoring and surveillance such as pulsed-field gel electrophoresis (PFGE) (22, 34).Initial isolation of V. parahaemolyticus is often conducted by culturing strains on thiosulfate citrate bile salts sucrose (TCBS) growth medium (15, 23). TCBS is used to selectively enrich for Vibrio spp. from cooccurring non-Vibrio strains; however, TCBS cannot differentiate V. parahaemolyticus from closely related species such as Vibrio harveyi and Vibrio campbellii. Additional molecular analyses are required to positively distinguish V. parahaemolyticus from other, closely related Vibrio species. These methods include group-specific PCR (4), multiplex PCR (38), multilocus sequence analysis (MLSA) (9, 17), comparative gene arrays (43), and whole-genome arrays (18). Often, several of these techniques are employed to distinguish V. parahaemolyticus from closely related Vibrio spp. and to provide greater resolution for discriminating among the pandemic clones (17, 18, 27). The development of a rapid method to distinguish V. parahaemolyticus from other Vibrio species including Vibrio pathogens would greatly aid the identification of strains involved in disease outbreaks when time is critical.Recent studies have shown that whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the rapid identification of bacteria including Streptococcus spp. (44), Salmonella strains (14), Mycobacterium spp. (35), Arthrobacter spp. (42), Listeria spp. (2), Burkholderia spp. (41), and other diverse nonfermenting clinical bacteria (12, 29). These studies have demonstrated the use of whole-cell MALDI-TOF MS analysis to generate highly reproducible and unique profiles to differentiate these bacterial strains at the species and subspecies levels. Whole-cell MALDI-TOF MS involves growing bacteria under standardized conditions and preparing cells for analysis by washing them to remove residual medium components, followed by resuspension of cells in a matrix that allows protein ionization. The cell-matrix suspension is then spotted onto a MALDI plate, each spot is ionized with a laser, and the ionizable proteins migrate based on their size resulting in the different peak sizes (kDa) in the MALDI-TOF MS spectra. Bacteria are typically grown overnight; however, the specific growth conditions and medium type must be determined and replicated to avoid condition-dependent differences in MADLI-TOF MS spectra (42). The method for preparation of the cells consists of only a few steps, and the protein ionization and generation of the spectra take several seconds. Whole-cell MALDI-TOF MS analysis can thus quickly provide accurate and reproducible generation of bacterial fingerprints that may be analyzed for the presence of biomarker peaks representative of a species or clonal group (2, 25, 35, 41, 44).In the current study, we have developed a method for whole-cell MALDI-TOF MS identification of V. parahaemolyticus. MALDI-TOF MS analysis was used to differentiate V. parahaemolyticus from nine other Vibrio spp. (V. campbellii, V. cholerae, V. fischeri, V. fluvialis, V. harveyi, V. vulnificus, V. alginolyticus, V. mimicus, and V. mediterranei) and to identify potential V. parahaemolyticus-specific biomarker peaks. The objectives of this study were to determine whether MALDI-TOF MS analysis is reliable for (i) distinguishing V. parahaemolyticus from closely related Vibrio spp. and (ii) detecting variation among the V. parahaemolyticus pandemic clones. Furthermore, we analyzed whether strains that have undergone single gene deletions will have unique fingerprints resulting from changes in their ionizable proteins. This is the first study to use whole-cell MALDI-TOF MS analysis to generate reproducible and unique fingerprints that may be used to rapidly identify Vibrio spp. and to distinguish V. parahaemolyticus from related vibrios.     

Identification of Putative Biomarkers for Toluene-Degrading Burkholderia and Pseudomonads by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and Peptide Mass Fingerprinting

The use of peptide mass fingerprinting with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was demonstrated to identify and phenotypically characterize toluene-degrading bacteria via biomarkers of degradation and taxonomical classification. Pseudomonas putida F1, P. mendocina KR1, and Burkholderia sp. JS150 were grown on toluene, extracted, electrophoretically separated, and analyzed by MALDI-TOF MS. Catabolic enzymes were identified and results substantiated using tandem MS.     

Rapid Identification and Typing of Listeria Species by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Listeria monocytogenes is a food-borne pathogen that is the causative agent of human listeriosis, an opportunistic infection that primarily infects pregnant women and immunologically compromised individuals. Rapid, accurate discrimination between Listeria strains is essential for appropriate therapeutic management and timely intervention for infection control. A rapid method involving matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) that shows promise for identification of Listeria species and typing and even allows for differentiation at the level of clonal lineages among pathogenic strains of L. monocytogenes is presented. A total of 146 strains of different Listeria species and serotypes as well as clinical isolates were analyzed. The method was compared with the pulsed-field gel electrophoresis analysis of 48 Listeria strains comprising L. monocytogenes strains isolated from food-borne epidemics and sporadic cases, isolates representing different serotypes, and a number of Listeria strains whose genomes have been completely sequenced. Following a short inactivation/extraction procedure, cell material from a bacterial colony was deposited on a sample target, dried, overlaid with a matrix necessary for the MALDI process, and analyzed by MALDI-TOF MS. This technique examines the chemistry of major proteins, yielding profile spectra consisting of a series of peaks, a characteristic “fingerprint” mainly derived from ribosomal proteins. Specimens can be prepared in a few minutes from plate or liquid cultures, and a spectrum can be obtained within 1 minute. Mass spectra derived from Listeria isolates showed characteristic peaks, conserved at both the species and lineage levels. MALDI-TOF MS fingerprinting may have potential for Listeria identification and subtyping and may improve infection control measures.     

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry for Identification of Microorganisms in Clinical Urine Specimens after Two Pretreatments

Rapid identification of microorganisms in urine is essential for patients with urinary tract infections (UTIs). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been proposed as a method for the direct identification of urinary pathogens. Our purpose was to compare centrifugation-based MALDI-TOF MS and short-term culture combined with MALDI-TOF MS for the direct identification of pathogens in urine specimens. We collected 965 urine specimens from patients with suspected UTIs, 211/965 isolates were identified as positive by conventional urine culture. Compared with the conventional method, the results of centrifugation-based MALDI-TOF MS were consistent in 159/211 cases (75.4%), of which 135/159 (84.9%) had scores ≥ 2.00; 182/211 cases (86.3%) were detected using short-term culture combined with MALDI-TOF MS, of which 153/182 (84.1%) had scores ≥ 2.00. There were no apparent differences among the three methods (p = 0.135). MALDI-TOF MS appears to accelerate the microbial identification speed in urine and saves at least 24 to 48 hours compared with the routine urine culture. Centrifugation-based MALDI-TOF MS is characterized by faster identification speed; however, it is substantially affected by the number of bacterial colonies. In contrast, short-term culture combined with MALDI-TOF MS has a higher detection rate but a relatively slow identification speed. Combining these characteristics, the two methods may be effective and reliable alternatives to traditional urine culture.     

Characterization of Cryptosporidium parvum by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) was used to investigate whole and freeze-thawed Cryptosporidium parvum oocysts. Whole oocysts revealed some mass spectral features. Reproducible patterns of spectral markers and increased sensitivity were obtained after the oocysts were lysed with a freeze-thaw procedure. Spectral-marker patterns for C. parvum were distinguishable from those obtained for Cryptosporidium muris. One spectral marker appears specific for the genus, while others appear specific at the species level. Three different C. parvum lots were investigated, and similar spectral markers were observed in each. Disinfection of the oocysts reduced and/or eliminated the patterns of spectral markers.     

Nonradioactive Phosphopeptide Assay by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry: Application to Calcium/Calmodulin-Dependent Protein Kinase II

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) was used to quantify the phosphopeptide produced by calcium/calmodulin-dependent protein kinase II (CaMK II). MALDI-TOF measurements were performed in a linear and positive ion mode with delayed extraction excited at various laser powers and at different sampling positions, i.e., different loci of laser illumination. We find that the ratio of the peak area of the substrate (S) to that of its monophosphorylated form (SP) for a given mixture is constant, independent of the laser powers and/or of the sample loci illuminated by the laser. We also find that the fraction of phosphorylation determined by MALDI-TOF, orf_MALDI-TOF, is proportionally smaller than that determined by HPLC, orf_HPLC; the ratiof_MALDI-TOF/f_HPLCwas 0.797 ± 0.0229 (99% confidence limit,n= 7) for a 30-mer peptide substrate used in this study. A low mass gate, which turns off the detector temporarily, improved the ratiof_MALDI-TOF/f_HPLCto 0.917 ± 0.0184 (99% confidence limit,n= 7). Our interpretation of this result is that the reduction of the phosphopeptide peak in the MALDI-TOF measurement is likely to be caused by a temporal loss of detector function rather than by a lower efficiency of ionization for the phosphopeptide compared with its parent species. In these measurements the experimental errors, up to the 50% phosphorylation state, were less than 5%. After an adjustment made based on thef_MALDI-TOF/f_HPLCratio of 0.917, MALDI-TOF gave an accurate measurement for the kinetics of the CaMK II phosphorylation reaction. Since only a small volume of the reaction mixture, typically containing 3 to 50 pmol of substrate, is required for the MALDI-TOF measurement, this method can be adapted to a nonradioactive microscale assay for CaMK II and also for other protein kinases.     

Rapid Characterization of Microalgae and Microalgae Mixtures Using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS)

Current molecular methods to characterize microalgae are time-intensive and expensive. Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) may represent a rapid and economical alternative approach. The objectives of this study were to determine whether MALDI-TOF MS can be used to: 1) differentiate microalgae at the species and strain levels and 2) characterize simple microalgal mixtures. A common protein extraction sample preparation method was used to facilitate rapid mass spectrometry-based analysis of 31 microalgae. Each yielded spectra containing between 6 and 56 peaks in the m/z 2,000 to 20,000 range. The taxonomic resolution of this approach appeared higher than that of 18S rDNA sequence analysis. For example, two strains of Scenedesmus acutus differed only by two 18S rDNA nucleotides, but yielded distinct MALDI-TOF mass spectra. Mixtures of two and three microalgae yielded relatively complex spectra that contained peaks associated with members of each mixture. Interestingly, though, mixture-specific peaks were observed at m/z 11,048 and 11,230. Our results suggest that MALDI-TOF MS affords rapid characterization of individual microalgae and simple microalgal mixtures.     

Characterization of Ribosomes from Dormant Spores of Bacillus cereus

Characterization of ribosomes from dormant spores and vegetative cells of Bacillus cereus strain T has been carried out. Polyuridylic acid binding activity, ribonuclease activity associated with ribosomes, thermal denaturation profile, and sedimentation coefficients are essentially identical for both ribosomal preparations. However, ribosomal protein content of dormant spore ribosomes is about 70% of that of vegetative ribosomes. Polyacrylamide gel electrophoresis of ribosomal proteins shows that some ribosomal proteins are missing from dormant spore ribosomes. Sucrose density gradient centrifugation of ribosomes shows the existence of defective ribosomal subunits, in addition to 30S and 50S subunits, in dormant spore ribosomes. These results indicate that the ribosomes from dormant spores are distinctively different from those of vegetative cells.     

Rapid Identification of the Foodborne Pathogen Trichinella spp. by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Human trichinellosis occurs through consumption of raw or inadequately processed meat or meat products containing larvae of the parasitic nematodes of the genus Trichinella. Currently, nine species and three genotypes are recognized, of which T. spiralis, T. britovi and T. pseudospiralis have the highest public health relevance. To date, the differentiation of the larvae to the species and genotype level is based primarily on molecular methods, which can be relatively time consuming and labor intensive. Due to its rapidness and ease of use a matrix assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF MS) reference spectra database using Trichinella strains of all known species and genotypes was created. A formicacid/acetonitrile protein extraction was carried out after pooling 10 larvae of each Trichinella species and genotype. Each sample was spotted 9 times using α-cyano 4-hydoxy cinnamic acid matrix and a MicroFlex LT mass spectrometer was used to acquire 3 spectra (m/z 2000 to 20000 Da) from each spot resulting in 27 spectra/species or genotype. Following the spectra quality assessment, Biotyper software was used to create a main spectra library (MSP) representing nine species and three genotypes of Trichinella. The evaluation of the spectra generated by MALDI-TOF MS revealed a classification which was comparable to the results obtained by molecular methods. Also, each Trichinella species utilized in this study was distinct and distinguishable with a high confidence level. Further, different conservation methods such as freezing and conservation in alcohol and the host species origin of the isolated larvae did not have a significant influence on the generated spectra. Therefore, the described MALDI-TOF MS can successfully be implemented for both genus and species level identification and represents a major step forward in the use of this technique in foodborne parasitology.     

Identification of Bacillus anthracis by Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Artificial Neural Networks

This report demonstrates the applicability of a combination of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and chemometrics for rapid and reliable identification of vegetative cells of the causative agent of anthrax, Bacillus anthracis. Bacillus cultures were prepared under standardized conditions and inactivated according to a recently developed MS-compatible inactivation protocol for highly pathogenic microorganisms. MALDI-TOF MS was then employed to collect spectra from the microbial samples and to build up a database of bacterial reference spectra. This database comprised mass peak profiles of 374 strains from Bacillus and related genera, among them 102 strains of B. anthracis and 121 strains of B. cereus. The information contained in the database was investigated by means of visual inspection of gel view representations, univariate t tests for biomarker identification, unsupervised hierarchical clustering, and artificial neural networks (ANNs). Analysis of gel views and independent t tests suggested B. anthracis- and B. cereus group-specific signals. For example, mass spectra of B. anthracis exhibited discriminating biomarkers at 4,606, 5,413, and 6,679 Da. A systematic search in proteomic databases allowed tentative assignment of some of the biomarkers to ribosomal protein or small acid-soluble proteins. Multivariate pattern analysis by unsupervised hierarchical cluster analysis further revealed a subproteome-based taxonomy of the genus Bacillus. Superior classification accuracy was achieved when supervised ANNs were employed. For the identification of B. anthracis, independent validation of optimized ANN models yielded a diagnostic sensitivity of 100% and a specificity of 100%.Members of the genus Bacillus are rod-shaped bacteria that exhibit catalase activity and can be characterized as endospore-forming obligate or facultative aerobes. The genus Bacillus contains two important groups of bacteria named after B. subtilis and B. cereus. The best-characterized member of the former group is B. subtilis, a renowned model organism for genetic research. Other group members, like B. pumilis, B. licheniformis, B. atrophaeus, and B. amyloliquefaciens, exhibit a high degree of phenotypic similarity and are thus not easily distinguishable (15).The B. cereus group comprises a number of closely related bacteria, some of which interfere with human health. Bacteria classified as B. cereus are occasionally associated with food poisoning (16, 28), while B. thuringiensis is primarily an insect pathogen because of its ability to produce toxins that have been widely used for the biocontrol of insect pests (28, 30). A third member of the B. cereus group, B. anthracis, is the causative agent of anthrax and is highly relevant to human and animal health. Other members of the B. cereus group are B. mycoides, B. pseudomycoides, and B. weihenstephanensis (4, 15).B. anthracis is a possible agent in biological warfare and bioterrorism. Its applicability as a biological warfare agent was made apparent by an accidental release from a Soviet military facility in Sverdlovsk (1, 10). Also, the well-publicized mailing of B. anthracis spores in the United States, which caused 18 confirmed cases of cutaneous and inhalational anthrax and an additional 4 suspected cases of cutaneous anthrax (3, 22), demonstrated that B. anthracis may become a threat from terrorist groups (10).Rapid detection of B. anthracis may be challenging because of its great genetic similarity to other species of the B. cereus group (10) and the difficulties of phenotypic differentiation of B. cereus group members (15). There is some controversy in the literature regarding the taxonomy of the B. cereus group. Indeed, some authors state that B. anthracis, B. cereus, and B. thuringiensis are one species with various virulence plasmids for the toxin pXO1 and the capsule pXO2 of B. anthracis and the insecticidal toxin of B. thuringiensis (10, 19). Other authors do not support this opinion and suggest the presence of even more species within the group (21).Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) intact-cell mass spectrometry (ICMS) has been suggested as a rapid, objective, and reliable technique for bacterial identification (8, 13, 23, 25, 38). As a proteomic technique, ICMS of whole bacterial cells, or cell lysates, relies on the reproducible detection of microbial protein patterns and thus delivers information complementary to genotypic or phenotypic test methods. With the pattern-matching approach, microbial identification is achieved by comparing experimental mass spectra with a collection of mass spectra of known organisms. This requires the compilation of large databases of bacterial reference spectra but has the advantage that an extensive knowledge of biomarker identities is not required. Another advantage of the pattern-matching approach is that genus- and species-specific procedures or consumables are not required, i.e., the same methodology can in principle be applied to all kinds of microorganisms (multiplex advantage).It is thus believed that ICMS offers the possibility to systematically investigate the diversity of bacterial subproteomes, complementing existing methodologies of bacterial characterization. This potential and the need for a rapid, objective, and reliable microbial identification technique that does not rely on nucleic acid detection and the availability of an MS-compatible inactivation protocol for highly pathogenic biosafety level 3 microorganisms and bacterial endospores (26) prompted us to systematically study the MALDI-TOF MS profiles of Bacillus strains and to establish a database of bacterial mass spectra. In the present work, we describe strategies of spectral analysis that allow the identification and validation of group- and species-specific sets of biomarkers. Using unsupervised hierarchical cluster analysis (UHCA) and supervised artificial neural network (ANN) analysis, we also demonstrate how microbial spectra can be employed to establish an MS-based methodology for rapid, objective, and reliable identification of the target species, B. anthracis.     

Characterization of Bacteria by Particle Beam Mass Spectrometry

A technique is described for detecting and characterizing bacteria on a single-particle basis by mass spectrometry. The method involves generation of a particle beam of single whole cells which are rapidly volatilized and ionized in vacuum in the ion source of a quadrupole mass spectrometer. The particle beam can be generated, with minimal sample handling, from a naturally occurring aerosol or from a solution of bacteria that can be dispersed as an aerosol. The mass spectrum is generated by successively measuring the average intensities of different mass peaks. The average intensity is obtained by measuring the ion intensity distribution at the particular mass (m/e) for ion pulses from more than 1,000 bacteria particles. Bacillus cereus, Bacillus subtilis, and Pseudomonas putida samples were analyzed to test the capability of the instrument for differentiating among species of bacteria. Significant ion-intensity information was produced over the m/e range of 50 to 300, an improvement over previous pyrolysis-mass spectrometry results. The complex mass spectra contained a few unique peaks which could be used for the differentiation of the bacteria. A statistical analysis of the variations in peak intensities among the three bacteria provided a quantitative measure of the reproducibility of the instrument and its ability to differentiate among bacteria. The technique could lead to a new rapid method for the analysis of microorganisms and could be used for the detection of airborne pathogens on a continuous, real-time basis.     

Application of Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Rapid Identification of Neisseria Species

Atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry (AP-MALDI MS) was applied to develop a proteomics-based method to detect and identify Neisseria species. Heat-inactivated clinical isolate cell suspensions of Neisseria gonorrhoeae and strains belonging to five serogroups (A, B, C, W135, and Y) of Neisseria meningitidis were subjected to on-probe protein/peptide extraction and tryptic digestion followed by AP-MALDI tandem MS (MS/MS)-based proteomic analysis. Amino acid sequences derived from three protonated peptides with m/z values of 1743.8, 1894.8, and 1946.8 were identified by AP-MALDI MS/MS and MASCOT proteome database search analysis as belonging to neisserial acyl carrier protein, neisserial-conserved hypothetical protein, and neisserial putative DNA binding protein, respectively. These three peptide masses can thus be potential biomarkers for neisserial species identification by AP-MALDI MS.