Detection of Microorganisms using MALDI and ion mobility mass spectrometry

Matrix-assisted laser ablation desorption ionization MALDI and ion mobility (IM) MALDI mass spectrometry (MS) were used for the detection and identification of microorganisms. MALDI MS is an analytical tool that separates ions by their mass-to-charge ratio (m/z) and is routinely used for bioanalysis because of its sensitivity, selectivity, general applicability, and tolerance to impurities. Ion mobility is a gas phase technique that separates ions based on their charge and collision cross-section. In this research, MALDI-TOF MS and MALDI-IM-TOF MS analysis were conducted in parallel to assess the effectiveness of MALDI-IM-TOF MS for microorganism identification. Whole cell bacteria Escherichia coli strain W 9637 and Bacillus subtilis 6633 were prepared and analyzed using both MALDI-TOF MS and MALDI-IM-TOF MS. The signals from both analysis methods were identified using a microbial database. Vacuum ultraviolet (VUV) post-ionization MALDI-IM-TOF MS was also used and additional peaks that could not be detected using MALDI-TOF MS and MALDI-IM-TOF MS were observed from B. subtilis. MALDI MS was used in combination with mass spectral fingerprinting software for the identification of whole cell bacteria in the presence of potential environmental interferants. Whole bacteria were analyzed in the presence of fumed silica, bentonite, and pollen from Juglans nigra (black walnut) at various mass ratios. The effect of the interferants on the identifications of bacteria at the genus and species level was evaluated using the bacteria fingerprinting software MB. The results showed that correct species identification for E. coli 35218, could be determined with fumed silica, bentonite, and pollen at a mass ratio of 1:1; whereas, at the same mass ratio, with diesel particulate, only genus identification could be made. Species identification for E. aerogenes 13048 with fumed silica and pollen at a mass ratio of 1:1 was achieved. Genus identification was determined for E.aerogenes with bentonite and diesel particulate. As the mass ratio of the interferant increased, the likelihood of species identification decreased with the exception of E. aerogenes with fumed silica and pollen. Under ambient conditions, laser ablation sample transfer using a mid-infrared laser at 2.94 ƒÝm was used to ablate gram-negative E. coli 35218 and gram-positive B. cereus 11178 bacterial colony particulate from a petri dish into a solvent droplet suspended above the petri dish. The solvent droplet containing the captured material was then transferred to a nanostructured-assisted laser desorption ionization (NALDI) target for analysis on a matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometer (MS). Several peaks that were observed in the NALDI spectra of both gram-negative and gram-positive correspond to phospholipid classes, phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). Additional phospholipids diglycosyldiglyceride (DGDG), triacylglyceride (TAG) and a lipopeptide, which are typically found in gram-positive bacteria were observed in the NALDI spectrum of B. cereus. Using LAST NALDI, phospholipids could be identified from both bacterial species without any sample pretreatment

Avaliação da técnica de MALDI-TOF MS no laboratório de microbiologia

Rapid identification of microorganisms by the clinical microbiology laboratory is of crucial importance for optimal patients’ management and treatment. In general, bacterial identification by conventional methods requires 18-24 hours for colony isolation and at least 24 additional hours for species identification. New technologies in microbiology have focused on the rapid diagnosis of bloodstream infections, since they are associated with high morbidity and mortality rates.A rápida identificação de microrganismos no laboratório de microbiologia clínica é de extrema importância para direcionar o manejo e o tratamento de pacientes. Geralmente, a identificação bacteriana por métodos bioquímicos convencionais necessita de 18 a 24 horas para o crescimento e o isolamento da colônia bacteriana e, pelo menos, 24 horas adicionais para a identificação da espécie. Novas tecnologias em microbiologia têm focado no desenvolvimento de métodos relacionados com o diagnóstico rápido das infecções da corrente sanguínea, uma vez que essas infecções são associadas à alta morbimortalidade.UNIFESPUNIFESP Infectious Disease Department ALERTA laboratoryNational Council for Scientific and Technological DevelopmentBrazilian Society of Infectious Diseases Bacteriology CommitteeUNIFESP, Infectious Disease Department ALERTA laboratorySciEL

Matrix-assisted laser desorption/ionization mass spectrometry for the analysis of collected bioaerosols

The goal of this dissertation was to demonstrate collection, detection and identification of microorganisms from bioaerosols using offline matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the analysis of proteins. MALDI-MS intact bacteria techniques were adapted for use with an orthogonal MALDI quadrupole-time-of-flight mass spectrometer. Results indicate the instrument is capable of analyzing intact whole-cells. The first phase was to evaluate three bioaerosol samplers, an Andersen N6 single-stage impactor (AI), a cyclone impactor, and a vacuum filter system. The samplers collected test bioaerosols using a constructed bioaerosol exposure chamber (BEC). The BEC allowed all three samplers to operate in parallel. Each sampler demonstrated the ability to successfully collect and detect the test bioaerosol by offline MALDI-MS. Using the TOF-MS spectra from impacted bacteria, the Expert Protein Analysis System\u27s (ExPASy) sequence retrieval system (SRS) was used to search the SWISS-PROT database. A total of 19 unique proteins were identified for E. coli,8 for B. Thuringiensis, and 6 for B. subtilis. Subsequently, cytochrome c and E. coli samples were proteolyzed in situ using trypsin and CNBr. The digestions were done using removable mini-wells. The mini-wells were placed on top of collected spots on the MALDI target and served as a mini chemical reactor for digestion. Using the TOF-MS spectra of the digested samples, peptide mass mapping was done using the MASCOT search engine. A progressive reductive iterative search mapping (PRISM) technique was used in order to assist in optimizing protein matches from E. coli. In this approach, four of seven iterations produced protein matches. To determine the suitability of MS/MS techniques for use with in situ digests, selected fragments from the cytochrome c and E. coli digests was done. MS/MS was successful for cytochrome c, but was unable to produce spectra for E. coli

Applications of MALDI-TOF mass spectrometry in clinical diagnostic microbiology.

Until recently, microbial identification in clinical diagnostic laboratories has mainly relied on conventional phenotypic and gene sequencing identification techniques. The development of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) devices has revolutionized the routine identification of microorganisms in clinical microbiology laboratories by introducing an easy, rapid, high throughput, low-cost, and efficient identification technique. This technology has been adapted to the constraint of clinical diagnostic laboratories and has the potential to replace and/or complement conventional identification techniques for both bacterial and fungal strains. Using standardized procedures, the resolution of MALDI-TOF MS allows accurate identification at the species level of most Gram-positive and Gram-negative bacterial strains with the exception of a few difficult strains that require more attention and further development of the method. Similarly, the routine identification by MALDI-TOF MS of yeast isolates is reliable and much quicker than conventional techniques. Recent studies have shown that MALDI-TOF MS has also the potential to accurately identify filamentous fungi and dermatophytes, providing that specific standardized procedures are established for these microorganisms. Moreover, MALDI-TOF MS has been used successfully for microbial typing and identification at the subspecies level, demonstrating that this technology is a potential efficient tool for epidemiological studies and for taxonomical classification

Applications of MALDI-TOF mass spectrometry in clinical diagnostic microbiology

Until recently, microbial identification in clinical diagnostic laboratories has mainly relied on conventional phenotypic and gene sequencing identification techniques. The development of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) devices has revolutionized the routine identification of microorganisms in clinical microbiology laboratories by introducing an easy, rapid, high throughput, low-cost, and efficient identification technique. This technology has been adapted to the constraint of clinical diagnostic laboratories and has the potential to replace and/or complement conventional identification techniques for both bacterial and fungal strains. Using standardized procedures, the resolution of MALDI-TOF MS allows accurate identification at the species level of most Gram-positive and Gram-negative bacterial strains with the exception of a few difficult strains that require more attention and further development of the method. Similarly, the routine identification by MALDI-TOF MS of yeast isolates is reliable and much quicker than conventional techniques. Recent studies have shown that MALDI-TOF MS has also the potential to accurately identify filamentous fungi and dermatophytes, providing that specific standardized procedures are established for these microorganisms. Moreover, MALDI-TOF MS has been used successfully for microbial typing and identification at the subspecies level, demonstrating that this technology is a potential efficient tool for epidemiological studies and for taxonomical classificatio

The Effects of Fungal Pigments on Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectral Peak Formation

Fungi are ubiquitous microorganisms that commonly cause diseases with high mortality rates in immunocompromised hosts. In order to successfully treat the infection and subsequently decrease mortality rates, it is necessary to rapidly identify the causal organism(s) and promptly begin the proper treatment. Common identification methods, such as viable culture characterization, are often time consuming or not species-specific, as is the case with many molecular or immunodiagnostic assays. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used to discriminate between many clinically relevant microorganisms. Recently, MALDI-TOF MS has been used to discriminate between monilaceous fungal species, however, darkly pigmented fungi yield poor mass spectra with only a few peaks of low relative abundance. Darkly pigmented fungi are of interest because they are common indoor contaminants, and frequently a cause of disease in immunocompromised patients. In this study, the effect of dark fungal pigments on the ionization of standard peptide and protein solutions during MALDI-TOF MS is investigated. Solutions of human serum albumin and angiotensin II were spiked with varying concentrations of both synthetic melanin and fungal pigments extracted from Aspergillus niger, A. fumigatus, and A. terreus. Serum albumin and angiotensin II [M+H]+ signals were suppressed in a concentration dependent manner. Examination of the MALDI sample deposit under magnification showed significant heterogeneity, with regions of highly concentrated pigment appearing black. MALDI-TOF mass spectra acquired from darkly pigmented regions of the sample deposit yielded poor or no [M+H]+ ion signal from the standard. In contrast, nonpigmented regions within the sample deposit and liquid hyphal negative control extracts of A. niger, A. fumigatus, and A. terreus produced no such inhibition. Gas chromatography analysis of each fungal pigment extract revealed the presence of putative cell wall components including aliphatic chains and fatty acids. Field emission scanning electron microscopy (FESEM) showed A. niger and A. fumigatus extracts retain original spore morphology, while the A. terreus extract was homogeneous and morphologically similar to synthetic melanin. This study demonstrates that synthetic and fungal pigmentation derived from darkly pigmented fungi inhibits the desorption/ionization process during MALDI-TOF MS, however these fungi may be successfully analyzed by MALDI-TOF MS when culture methods that suppress pigment expression are employed

Proteomics boosts translational and clinical microbiology

The application of proteomics to translational and clinical microbiology is one of the most advanced frontiers in the management and control of infectious diseases and in the understanding of complex microbial systems within human fluids and districts. This new approach aims at providing, by dedicated bioinformatic pipelines, a thorough description of pathogen proteomes and their interactions within the context of human host ecosystems, revolutionizing the vision of infectious diseases in biomedicine and approaching new viewpoints in both diagnostic and clinical management of the patient.Indeed, in the last few years, many laboratories have matured a series of advanced proteomic applications, aiming at providing individual proteome charts of pathogens, with respect to their morph and/or cell life stages, antimicrobial or antimycotic resistance profiling, epidemiological dispersion. Herein, we aim at reviewing the current state-of-the-art on proteomic protocols designed and set-up for translational and diagnostic microbiological purposes, from axenic pathogens' characterization to microbiota ecosystems' full description. The final goal is to describe applications of the most common MALDI-TOF MS platforms to advanced diagnostic issues related to emerging infections, increasing of fastidious bacteria, and generation of patient-tailored phylotypes. This article is part of a Special Issue entitled: Trends in Microbial Proteomics. © 2013 The Authors

Identification of Brucella by MALDI-TOF Mass Spectrometry. Fast and Reliable Identification from Agar Plates and Blood Cultures

BACKGROUND: MALDI-TOF mass spectrometry (MS) is a reliable method for bacteria identification. Some databases used for this purpose lack reference profiles for Brucella species, which is still an important pathogen in wide areas around the world. We report the creation of profiles for MALDI-TOF Biotyper 2.0 database (Bruker Daltonics, Germany) and their usefulness for identifying brucellae from culture plates and blood cultures. METHODOLOGY/PRINCIPAL FINDINGS: We created MALDI Biotyper 2.0 profiles for type strains belonging to B. melitensis biotypes 1, 2 and 3; B. abortus biotypes 1, 2, 5 and 9; B. suis, B. canis, B ceti and B. pinnipedialis. Then, 131 clinical isolates grown on plate cultures were used in triplicate to check identification. Identification at genus level was always correct, although in most cases the three replicates reported different identification at species level. Simulated blood cultures were performed with type strains belonging to the main human pathogenic species (B. melitensis, B. abortus, B. suis and B. canis), and studied by MALDI-TOF MS in triplicate. Identification at genus level was always correct. CONCLUSIONS/SIGNIFICANCE: MALDI-TOF MS is reliable for Brucella identification to the genus level from culture plates and directly from blood culture bottles

Application of MALDIs-MS to Identification of Phytoplankton in Ballast Water

Non-native invasive species are increasingly evident in marine and estuarine environments, largely because of the intake and release of ballast water from sea vessels. Innovative methods are needed to quickly and accurately detect and speciate non-native and/or harmful phytoplankton in ballast water. Recent advances in ionization techniques such as matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) have allowed detection of intact biomolecules within ballast samples. Statistically-based algorithms are used to discern fingerprints of phytoplankton species and to discern individual species from mixtures. MALDI-MS is particularly attractive for field applications because of the speed of analysis, minimal liquids/consumables required, and femtomole (10-15) sensitivity. The objective of this project was to develop a rapid assay technique that was less time-consuming than more traditional methods of microorganism species identification in ballast water samples. Specific objectives included: (1) modifying MALDI-MS bacterial identification techniques for the analysis of phytoplankton, (2) determining the uniqueness of phytoplankton MALDI-MS fingerprints with a limited subset of phytoplankton, and (3) initiating evaluations of known phytoplankton identification in spiked environmental/ballast water samples