Rapid characterization of <i>Bacillus</i> spores targeting species‐unique peptides produced with an atmospheric pressure matrix‐assisted laser desorption/ionization source

Pribil, Patrick; Patton, Elizabeth; Black, Gavin E.; Doroshenko, Vladimir M.; Fenselau, Catherine

doi:10.1002/jms.816

Cited by 49 publications

(39 citation statements)

References 38 publications

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“…The SASPs found in B. anthracis spores were used as B. anthracis-specific protein markers [29]. This marker has been found to be insufficient to discriminate amongst closely related organisms [30]; however, realtime PCR consists of confirmation of three targets, namely the SASP, and plasmid targets, which improves the specificity of the test.…”

Section: Discussionmentioning

confidence: 99%

Polyphasic characterization of Bacillus species from anthrax outbreaks in animals from South Africa and Lesotho

Lekota

Hassim

Mafofo

et al. 2016

J Infect Dev Ctries

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Introduction: Bacillus anthracis is the causative agent of anthrax, a disease endemic in regions of Northern Cape Province and Kruger National Park of South Africa. Accurate identification of virulent B. anthracis is essential but challenging due to its close relationship with other members of B. cereus group. This study characterized B. anthracis and Bacillus species that were recovered from animals and the environment where animals died of anthrax symptoms in southern Africa using a polyphasic approach. Methodology: For this purpose, 3 B. anthracis and 10 Bacillus isolates were subjected to microbiology tests, BiologOmniLog identification system (Biolog), 16S ribosomal RNA (rRNA) sequence analysis, polymerase chain reaction (PCR) detection of protective antigen (pag) and capsule (cap) regions, and real-time PCR using hybridization probes targeting chromosomal, pag, and capC genes. Results: The Bacillus isolates were non-hemolytic, non-motile, and susceptible to penicillin, which is typical of B. anthracis, but resistant to gamma phage, unlike typical B. anthracis. The Biolog system and 16S rRNA gene sequence analysis identified most of the Bacillus isolates as B. endophyticus (7 of 10). Conventional PCR revealed that most of the Bacillus isolates contained capBCA gene regions. This highlights the limitation of the specificity of conventional PCR and the fact that the real-time PCR is more specific and reliable for anthrax diagnosis. Conclusions: Real-time PCR, 16S rRNA sequencing, and confirmatory microbiology tests including phage resistance distinguished Bacillus isolates from B. anthracis in this study. Identification of B. anthracis should be done using a polyphasic approach.

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Section: Discussionmentioning

confidence: 99%

Polyphasic characterization of Bacillus species from anthrax outbreaks in animals from South Africa and Lesotho

Lekota

Hassim

Mafofo

et al. 2016

J Infect Dev Ctries

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“…To date, MALDI-MS proteomics methods focused on B. anthracis detection have targeted chromosomally encoded proteins [13][14][15]. Unfortunately, nearly all chromosomally encoded proteins of B. anthracis have high sequence conservation between closely related species (B. anthracis, B. thuringiensis, and B. cereus), which hinders species-level identification.…”

Section: Discussionmentioning

confidence: 99%

“…MALDI-MS can be applied to microorganism identification by detecting proteins expressed by the microorganism, but the proteins must be species-specific. However, the level of specificity of MALDI-MS techniques has been limited by extremely high amino acid sequence conservation of chromosomally encoded proteins between closely related species [13][14][15]. This problem is extremely prevalent for B. anthracis due to a high abundance of closely related species (B. thuringiensis and B. cereus) in the environment.…”

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confidence: 99%

Targeted proteomics approach to species-level identification of Bacillus thuringiensis spores by AP-MALDI-MS

Nguyen

Russell

2010

J. Am. Soc. Mass Spectrom.

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Anthrax infections progress at a rapid pace, making rapid detection methods of utmost importance. MALDI-MS proteomics methods focused on Bacillus anthracis detection have targeted chromosomally encoded proteins, which are highly conserved between closely related species, hindering species identification. Presented here is an AP-MALDI-MS method targeting plasmid-borne proteins from Bacillus spores for species-level identification. A bioinformatics analysis revealed that 60.3% and 75.4% of tryptic peptides from plasmid-borne proteins of B. anthracis and B. thuringiensis were species-specific, respectively. Reported here is a method in which plasmid-borne ␦-endotoxins were extracted directly from B. thuringiensis spores in 100 mM KOH. The pH was then adjusted to 8 and a 5-min trypsin digestion was performed on the extracted proteins. The resulting tryptic peptides were analyzed by AP-MALDI-MS/MS, which produced a definitive identification the B. thuringiensis speciesspecific Cry1Ab protein with a MASCOT score of 278 and expect value of 7.5 ϫ 10 Ϫ23. This method has demonstrated the detection and identification of B. thuringiensis spores at the species level following a 5-min trypsin digestion. The challenges in applying a similar approach to the detection of plasmid-borne protein toxins from B. anthracis are also discussed. (J Am Soc Mass Spectrom 2010, 21, 993-1001

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“…Mass spectrometry recently has been utilized as a tool for rapid identification of microorganisms in a variety of settings (6,12,17), and it is regarded as an important technique for the detection of bioterrorism agents, particularly in air (10,24). However, use of MS for identification of viruses in complex matrixes is still in its infancy.…”

Section: Discussionmentioning

confidence: 99%

Detection of Norovirus Capsid Protein in Authentic Standards and in Stool Extracts by Matrix-Assisted Laser Desorption Ionization and Nanospray Mass Spectrometry

Colquhoun

Schwab

Cole

et al. 2006

Appl Environ Microbiol

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Mass spectrometry (MS) represents a rapid technique for the identification of microbial monocultures, and its adaptation to the detection of pathogens in real-world samples is a public health and homeland security priority. Norovirus, a leading cause of gastroenteritis in the world, is difficult to monitor because it cannot be cultured outside the human body. The detection of norovirus capsid protein was explored using three common MS-based methods: scanning of intact proteins, peptide mass fingerprinting, and peptide sequencing. Detection of intact target protein was limited by poor selectivity and sensitivity. Detection of up to 16 target peptides by peptide mass fingerprinting allowed for the reproducible and confident (P < 0.05) detection of the 56-kDa norovirus capsid protein in the range of 0.1 ؋ 10 ؊12 to 50 ؋ 10 ؊12 mol in authentic standards of recombinant norovirus virus-like particles (VLPs). To explore assay performance in complex matrixes, a non-gel-based, rapid method (2 to 3 h) for virus extraction from human stool was evaluated (72% ؎ 12% recovery), and additional analyses were performed on norovirus-free stool extracts fortified with VLPs. Whereas peptide mass fingerprinting was rendered impractical by sample interferences, peptide sequencing using nanospray tandem MS facilitated unambiguous identification of >250 fmol of capsid protein in stool extracts. This is the first report on MS-based detection of norovirus, accomplished by using structurally identical, noninfective VLPs at clinically relevant concentrations. It represents an important milestone in the development of assays for surveillance of this category B bioterrorism agent.

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Rapid characterization of Bacillus spores targeting species‐unique peptides produced with an atmospheric pressure matrix‐assisted laser desorption/ionization source

Cited by 49 publications

References 38 publications

Polyphasic characterization of Bacillus species from anthrax outbreaks in animals from South Africa and Lesotho

Polyphasic characterization of Bacillus species from anthrax outbreaks in animals from South Africa and Lesotho

Targeted proteomics approach to species-level identification of Bacillus thuringiensis spores by AP-MALDI-MS

Detection of Norovirus Capsid Protein in Authentic Standards and in Stool Extracts by Matrix-Assisted Laser Desorption Ionization and Nanospray Mass Spectrometry

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