Plamen A. Demirev

Genomic Signatures of Strain Selection and Enhancement in Bacillus atrophaeus var. globigii ,a Historical Biowarfare Simulant

Background Despite the decades-long use of Bacillus atrophaeus var. globigii (BG) as a simulant for biological warfare (BW) agents, knowledge of its genome composition is limited. Furthermore, the ability to differentiate signatures of deliberate adaptation and selection from natural variation is lacking for most bacterial agents. We characterized a lineage of BGwith a long history of use as a simulant for BW operations, focusing on classical bacteriological markers, metabolic profiling and whole-genome shotgun sequencing (WGS). Results Archival strains and two “present day” type strains were compared to simulant strains on different laboratory media. Several of the samples produced multiple colony morphotypes that differed from that of an archival isolate. To trace the microevolutionary history of these isolates, we obtained WGS data for several archival and present-day strains and morphotypes. Bacillus-wide phylogenetic analysis identified B. subtilis as the nearest neighbor to B. atrophaeus. The genome of B. atrophaeus is, on average, 86% identical to B. subtilis on the nucleotide level. WGS of variants revealed that several strains were mixed but highly related populations and uncovered a progressive accumulation of mutations among the “military” isolates. Metabolic profiling and microscopic examination of bacterial cultures revealed enhanced growth of “military” isolates on lactate-containing media, and showed that the “military” strains exhibited a hypersporulating phenotype. Conclusions Our analysis revealed the genomic and phenotypic signatures of strain adaptation and deliberate selection for traits that were desirable in a simulant organism. Together, these results demonstrate the power of whole-genome and modern systems-level approaches to characterize microbial lineages to develop and validate forensic markers for strain discrimination and reveal signatures of deliberate adaptation.

Establishing Drug Resistance in Microorganisms by Mass Spectrometry

AbstractA rapid method to determine drug resistance in bacteria based on mass spectrometry is presented. In it, a mass spectrum of an intact microorganism grown in drug-containing stable isotope-labeled media is compared with a mass spectrum of the intact microorganism grown in non-labeled media without the drug present. Drug resistance is determined by predicting characteristic mass shifts of one or more microorganism biomarkers using bioinformatics algorithms. Observing such characteristic mass shifts indicates that the microorganism is viable even in the presence of the drug, thus incorporating the isotopic label into characteristic biomarker molecules. The performance of the method is illustrated on the example of intact E. coli, grown in control (unlabeled) and 13C-labeled media, and analyzed by MALDI TOF MS. Algorithms for data analysis are presented as well.n Figureᅟ

Mass spectrometry for malaria diagnosis.

A physical method currently being developed for malaria parasite detection and diagnosis in blood is reviewed in this article. The method – direct laser desorption mass spectrometry – is based on the detection of heme (iron protoporphyrin) as a unique qualitative and quantitative molecular biomarker for malaria. In infected erythrocytes, the parasite sequesters heme in a molecular crystal (hemozoin) – a volume of highly concentrated and purified biomarker molecules. Laser desorption mass spectrometry detects only heme from hemozoin in parasite-infected blood, and not heme that is bound to hemoglobin or other proteins in uninfected blood samples. The method requires only a drop of blood with minimal sample preparation. Laser desorption mass spectrometry may become a rapid and high-throughput tool for specific and sensitive pan-malaria detection at levels below 10 parasites/μl of blood.

Enhanced in-source fragmentation in MALDI-TOF-MS of oligonucleotides using 1,5-diaminonapthalene.

The capability to rapidly and confidently determine or confirm the sequences of short oligonucleotides, including native and chemically-modified DNA and RNA, is important for a number of fields. While matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has been used previously to sequence short oligonucleotides, the typically low fragmentation efficiency of in-source or post-source decay processes necessitates the accumulation of a large number of spectra, thus limiting the throughput of these methods. Here we introduce a novel matrix, 1,5-diaminonapthalene (DAN), for facile in-source decay (ISD) of DNA and RNA molecular anions, which allows for rapid sequence confirmation. d-, w-, and y-series ions are prominent in the spectra, complementary to the (a-B)- and w- ions that are typically produced by MALDI post-source decay (PSD). Results are shown for several model DNA and RNA oligonucleotides, including combinations of DAN-induced fragmentation with true tandem TOF MS (MS/MS) for pseudo-MS3 and “activated-ion PSD.”

Ion Mobility Spectrometry - High Resolution LTQ-Orbitrap Mass Spectrometry for Analysis of Homemade Explosives

AbstractThe detailed chemical characterization of homemade explosives (HMEs) and other chemicals that can mimic or mask the presence of explosives is important for understanding and improving the performance of commercial instrumentation used for explosive detection. To that end, an atmospheric-pressure drift tube ion mobility spectrometry (IMS) instrument has been successfully coupled to a commercial tandem mass spectrometry (MS) system. The tandem MS system is comprised of a linear ion trap and a high resolution Orbitrap analyzer. This IMS-MS combination allows extensive characterization of threat chemical compounds, including HMEs, and complex real-world background chemicals that can interfere with detection. Here, the composition of ion species originating from a specific HME, erythritol tetranitrate, has been elucidated using accurate mass measurements, isotopic ratios, and tandem MS. Gated IMS-MS and high-resolution MS have been used to identify minor impurities that can be indicative of the HME source and/or synthesis route. Comparison between data obtained on the IMS/MS system and on commercial stand-alone IMS instruments used as explosive trace detectors (ETDs) has also been performed. Such analysis allows better signature assignments of threat compounds, modified detection algorithms, and improved overall ETD performance.n Graphical Abstractᅟ

Chapter 14 – Mass spectrometry of infectious pathogens

Publisher Summary nThe soft ionization mass spectrometry (MS) techniques such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) are making new inroads into a number of new MS applications in life sciences and medicine. The current paradigm for rapid MS identifications of pathogens relies on the detection and identification of unique biomarker molecules from experimental mass spectra. Structural elucidation of the unique chemical biomarkers from different organisms has been achieved by MS. The signature composition and abundances allowed taxonomic distinctions between the microorganisms to be made. MS is an emerging biosensor technology for diagnosis of infectious diseases with several practical advantages such as speed, sensitivity, and specificity. MS can be interfaced to a variety of sample collection and sample processing modules to allow versatile sampling from different environments. MS can be automated and it is also computer-friendly such as the latest developments in bioinformatics can be coupled to MS experimental data for robust pathogen diagnosis. Furthermore, MS instruments can be miniaturized and deployed in the field. This chapter discusses MALDI MS for rapid identification of intact Bacillus spore species, as well as LD MS for detection in blood of Plasmodium parasites (the causative agent of malaria).

Interaction of near-infrared femtosecond laser pulses with biological materials in water

Abstract. We study the effects of the interaction of 40-fs Ti-sapphire laser radiation at 800 nm with biological materials—proteins or intact Bacillus spore, dissolved or suspended in pure water, respectively. The estimated laser intensity at the target is 1013u2009u2009W/cm2. On the molecular level, oxidation of solvent-accessible parts of proteins has been observed even after a single femtosecond laser pulse, as demonstrated by mass spectrometry. A remarkable morphological effect of the femtosecond laser radiation is the complete disintegration of extremely refractive cells such as bacterial spores, evidenced in scanning electron micrographs. After 500 laser pulses, all suspended spores in the irradiated volume are completely destroyed, which makes them nonviable. Characteristic spore biomolecules, e.g., small acid-soluble spore proteins, are extensively oxidized after several laser pulses. In comparative studies, no effects have been observed when irradiating the same samples with 10-ns laser pulses at the same laser wavelength and fluence. We demonstrate that the laser power density (irradiance), resulting in different amounts of total deposited energy, determines the types of effects for femtosecond laser interactions with biological matter.