One of the possible usages of MALDI–TOF MS is identification of microorganisms. It is highly accurate and faster than other methods and suitable for a large area of microorganisms. But unfortunately cannot reliably differentiate some closely related species. Often, it is possible to detect pathogenic organisms and use their accurate determination for monitoring environment, food processing or clinical diagnostics. Data obtained from mass spectra are compared with library of microorganisms and result of this was agreement or disagreement of microorganisms with specific spectrum.

• Keywords
• identifikace bakterií,
• MeSH
• Bacteria * isolation & purification   MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods   MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

Úvod: Včasná a kauzální aplikace antibiotik u pacientů s pozitivní hemokulturou patří mezi základní předpoklady úspěšné léčby infekce. Izolace a následná identifikace bakterií z hemokultury klasickými (kultivačními) metodami však může trvat až několik dní. MALDI-TOF MS patří mezi metody, které umožňují rychlou identifikaci bakterií, a to nejen kultur z kultivačních médií, ale i přímo v klinickém materiálu. Metodika: Do studie byly zařazeny vzorky pozitivních hemokultur, které byly v letech 2016 až 2018 odebrány od pacientů Fakultní nemocnice Olomouc a následně byly vyšetřeny na Ústavu mikrobiologie Lékařské fakulty Univerzity Palackého v Olomouci. Vzorky pozitivních hemokultur byly zpracovány pomocí vlastního modifikovaného postupu, zahrnujícího odstranění krevních buněk pomocí centrifugace za nižších otáček. Následně byla pomocí centrifugace za vyšších otáček a promývání vzorku získána peleta, která byla testována pomocí MALDI-TOF MS. Výsledky: Ve studii bylo metodou přímé identifikace vyšetřeno celkem 110 pozitivních hemokultur. Správná identifikace na úrovni druhu byla vyšší u gramnegativních bakterií (88 %) v porovnání s grampozitivními bakteriemi (79 %) a také u nich bylo dosaženo vyšších hodnot identifikačního skóre. Hodnota identifikačního skóre vyšší nebo rovna než 2,0 byla u 62 % hemokultur obsahujících gramnegativní bakterie a 17 % hemokultur obsahujících bakterie grampozitivní. Hodnota identifikačního skóre v rozmezí 1,7–2,0 byla zjištěna u 21 % hemokultur gramnegativních a 33 % hemokultur s grampozitivními bakteriemi. Závěr: Přímá identifikace mikroorganismů z pozitivních hemokultur metodou MALDI-TOF MS umožňuje rychlejší diagnostiku a v důsledku zkrácení doby potřebné k dosažení výsledku identifikace patogenu může pozitivním způsobem významně ovlivnit antibiotickou léčbu pacientů.

Background: Early and causal administration of antibiotics in patients with a positive blood culture is an essential prerequisite for successful treatment of infection. However, isolation and subsequent identification of bacteria in a blood culture by classical (culture) methods may last several days. MALDI-TOF MS is a method allowing rapid identification of bacteria, not only cultures from culture media, but also directly in clinical specimens. Methods: The study included samples of positive blood cultures taken from patients in the University Hospital Olomouc between 2016 and 2018 and examined at the Department of Microbiology of the Faculty of Medicine, Palacký University Olomouc. Positive blood culture samples were processed using an in-house method involving the removal of blood cells by low-speed centrifugation. Subsequently, a pellet obtained by high-speed centrifugation and sample washing was tested by MALDI-TOF MS. Results: A total of 110 positive blood cultures were examined using the method of direct identification. At a species level, more Gramnegative bacteria (88 %) than Gram-positive bacteria (79 %) were correctly identified, with higher identification score values being obtained for the former. Identification score values of 2.0 or higher were found in 62 % of blood cultures containing Gram-negative bacteria and 17 % of blood cultures containing Gram-positive bacteria. Identification score values ranging from 1.7 to 2.0 were found in 21 % of Gram-negative blood cultures and 33 % of blood cultures containing Gram-positive bacteria. Conclusion: Direct identification of microorganisms from positive blood cultures using MALDI-TOF MS enables more rapid diagnosis. By reducing the time required to obtain the result of pathogen identification, it may positively affect the antibiotic treatment of patients.

• MeSH
• Bacteremia * diagnostic imaging   microbiology   MeSH
• Gram-Negative Bacteria isolation & purification   pathogenicity   MeSH
• Gram-Positive Bacteria isolation & purification   pathogenicity   MeSH
• Humans MeSH
• Sensitivity and Specificity MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods   instrumentation   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

Cíl práce: Ověřit možnosti metody MALDI TOF MS pro rychlou identifikaci vybraných původců bakteriálních zoonóz izolovaných Z různých druhů materiálů v reálných podmínkách rutinního provozu laboratoře. Materiál a metodika: Od srpna 2010 do dubna 2015 bylo metodou MALDI TOF MS na přístroji Microflex LT (Bruker Daltonics) provedeno celkem 4 174 identifikací vybraných původců bakteriálních zoonóz (Salmonella spp., Campylobacter jejuni, Campylobacter coli, Listeria monocytogenes, Yersinia enterocolitica, Yersinia pseudotuberculosis, Francisella tularensis, Brucella melitensis, Brucella suis a Cronobacter sakazakií). Bakteriální izoláty byly připraveny k testování jednoduchou přípravou vzorku pomocí zakápnutí bakteriální kultury na terčíku kovové destičky speciální matricí. Vyhodnocení výsledků proběhlo pomocí standardního protokolu programem MALDI Biotyper v provozních podmínkách. Výsledky: U 74,8 % testovaných izolátů sledovaných bakteriálních druhů bylo dosaženo hodnoty identifikačního skóre (IS) v rozmezí 2-3, což z hlediska interpretace výsledku představuje v praxi uspokojivý výsledek. Urychlení diagnostiky bakterií Campylobacter spp. a Listeria monocytogenes testováním suspektních kultur získaných přímo ze selektivně-diagnostických půd v těchto případech snížío identifikační skóre. Závěr: MALDI TOF MS je vhodná a rychlá metoda k identifikaci sledovaných původců bakteriálních zoonóz.

Objective: To verify whether the MALDI TOF MS method can be used for rapid identification of selected zoonotic bacterial pathogens isolated from various types of materials in the real conditions of routine laboratory work. Material and methods: Between August 2010 and April 2015, the Bruker's MALDI TOF MS system was used for 4,174 identifications of selected zoonotic bacterial pathogens (Salmonella spp., Campylobacter jejuni, Campylobacter coli. Listeria monocytogenes, Yersinia enterocolitica. Yersinia pseudotuberculosis, Francisella tularensis, brucella melitensis. Brucella suis and Cronobacter sakazakii). The samples were prepared for the test by simply mixing a bacterial culture with a matrix on a steel target plate. The results were evaluated with a standard protocol of the system using the MALDI Biotyper software under operating conditions. Results: In 74.8% of the tested isolates of the above bacterial species, the identification scores ranged between 2 and 3, which is satisfactory for result interpretation in routine practice. Acceleration of identification of Campylobacter spp. and Listeria monocytogenes by testing suspicious cultures obtained directly from selective-diagnostic media decreased the identification scores in these cases. Conclusion: MALDI TOF MS is a suitable and rapid method for identification of the selected zoonotic bacterial pathogens.

• MeSH
• Bacteriological Techniques methods   instrumentation   utilization   MeSH
• Clinical Laboratory Techniques MeSH
• Humans MeSH
• Ribosomal Proteins analysis   MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization * methods   utilization   MeSH
• Zoonoses * microbiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• Bacteria isolation & purification   MeSH
• Bacterial Infections diagnosis   MeSH
• Culture Techniques methods   MeSH
• Blood Culture methods   MeSH
• Humans MeSH
• Microbiological Techniques * methods   MeSH
• Urine microbiology   MeSH
• Cerebrospinal Fluid microbiology   MeSH
• Sepsis diagnosis   MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization * methods   instrumentation   utilization   MeSH
• Bacterial Typing Techniques * methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Review MeSH

• Publication type
• Meeting Abstract MeSH

Cíl práce: MALDI-TOF hmotnostní spektrometrie je v posledních letech široce zaváděna do diagnostických mikrobiologických laboratoří. Poskytuje levnou a rychlou metodu pro taxonomickou identifikaci bakterií a mikromycet. Kromě těchto aplikací je používána i pro detekci mechanismů antibiotické rezistence. V budoucnu lze očekávat další rozšíření i pro jiné aplikace v mikrobiologii. Cílem této studie bylo validovat MALDI-TOF hmotnostní spektrometrii pro identifikaci mykobakterií. Materiál a metody: Do studie bylo zařazeno 30 izolátů Mycobacterium spp. izolovaných v laboratoři mykobakteriologie Fakultní nemocnice v Plzni. Druhová identifikace izolátů byla provedena biochemickými testy, genovými sondami a sekvenací genu pro 16S rRNA. Identifikace MALDI-TOF hmotnostní spektrometrií probíhala s využitím extrakce pomocí silikonových kuliček. Identifikace kmene sekvenací genu pro 16S rRNA byla považována za referenční metodu. Výsledky: Pomocí MALDI-TOF hmotnostní spektrometrie byly správně identifikovány všechny izoláty Mycobacterium spp. (hodnota skóre 1,461–2,168). Jednalo se o druhy Mycobacterium tuberculosis (n = 5), Mycobacterium kansasii (n = 5), Mycobacterium avium (n = 6), Mycobacterium intracelullare (n = 3), Mycobacterium xenopi (n = 3), Mycobacterium gordonae (n=1), Mycobacterium abscessus (n=1), Mycobacterium kumamotonense (n=2), Mycobacterium mantenii (n = 1), Mycobacterium lentiflavum (n = 1), Mycobacterium fortuitum (n = 1), Mycobacterium scrofulaceum (n = 1). Závěr: Identifikace hmotnostní spektrometrií je tedy vhodná k rutinní identifikaci Mycobacterium spp. v laboratořích, kde již je tato metoda zavedena pro konvenční identifikaci mikrobů.

Study objective: Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has recently been widely used in diagnostic microbiological laboratories. It is a cheap and rapid method for the identification of bacteria and micromycetes. Apart from this purpose, it is also used for the detection of antibiotic resistance mechanisms. It has the potential to be extended for other purposes in microbiology. The aim of this study was to validate MALDI-TOF MS for the identification of mycobacteria. Material and methods: Thirty isolates of Mycobacterium spp. isolated in the Laboratory of Mycobacteriology of the Plzeň University Hospital were included in the study. The isolates were identified to the species level using biochemical tests, gene probes, and sequencing of the gene encoding 16S rRNA. The identification by MALDI-TOF MS was performed with the use of silica beads. Strain identification by sequencing the gene encoding 16S rRNA was considered as the reference method. Results: MALDI-TOF MS correctly identified all isolates of Mycobacterium spp. (score range 1.461 – 2.168). The species identified were Mycobacterium tuberculosis (n= 5), Mycobacterium kansasii (n=5), Mycobacterium avium (n=6), Mycobacterium intracelullare (n=3), Mycobacterium xenopi (n=3), Mycobacterium gordonae (n=1), Mycobacterium abscessus (n=1), Mycobacterium kumamotonense (n=2), Mycobacterium mantenii (n=1), Mycobacterium lentiflavum (n=1), Mycobacterium fortuitum (n=1), and Mycobacterium scrofulaceum (n=1). Conclusion: MALDI-TOF MS is a suitable tool for the routine identification of Mycobacterium spp. in laboratories using this method for the conventional identification of microbes.

• MeSH
• Laboratories MeSH
• Humans MeSH
• Mycobacterium * genetics   isolation & purification   classification   MeSH
• Mycobacterium Infections * diagnosis   MeSH
• RNA, Ribosomal, 16S MeSH
• Sequence Analysis, DNA MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization * utilization   MeSH
• Bacterial Typing Techniques methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

Biochemical and serological profiles of isolates of Plesiomonas shigelloides were assayed using standard procedures in isolates from various clinical samples. Seventy-four isolates, including P. shigelloides type strain, were further characterized by MALDI-TOF MS using 3-methoxy-4-hydroxycinnamic acid as matrix. Multiple ions in the 3- to 12-kDa mass range were found in the spectra of each strain, from which the "species-identifying" unique biomarker ions were identified. After creating the species-specific patterns, a spectral database was generated for reliable, rapid, reproducible and accurate identification of Plesiomonas strains. The classical strain description (biochemical and serological) was thus complemented with the metabolic (proteomic) characterization.

• MeSH
• Bacteriological Techniques methods   MeSH
• Gram-Negative Bacterial Infections microbiology   MeSH
• Plesiomonas chemistry   immunology   isolation & purification   classification   MeSH
• Serotyping methods   MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods   MeSH

We report for the first time the efficient use of accelerated solvent extraction (ASE) for extraction of ricin to analytical purposes, followed by the combined use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and MALDI-TOF MS/MS method. That has provided a fast and unambiguous method of ricin identification for in real cases of forensic investigation of suspected samples. Additionally, MALDI-TOF MS was applied to characterize the presence and the toxic activity of ricin in irradiated samples. Samples containing ricin were subjected to ASE, irradiated with different dosages of gamma radiation, and analyzed by MALDI-TOF MS/MS for verification of the intact protein signal. For identification purposes, samples were previously subjected to SDS-PAGE, for purification and separation of the chains, followed by digestion with trypsin, and analysis by MALDI-TOF MS/MS. The results were confirmed by verification of the amino acid sequences of some selected peptides by MALDI-TOF MS/MS. The samples residual toxic activity was evaluated through incubation with a DNA substrate, to simulate the attack by ricin, followed by MALDI-TOF MS/MS analyses.

• MeSH
• Acetone chemistry   MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Hexanes chemistry   MeSH
• Peptides analysis   chemistry   MeSH
• Ricin analysis   chemistry   MeSH
• Solvents chemistry   MeSH
• Amino Acid Sequence MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
• Tandem Mass Spectrometry MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Dickeya and Pectobacterium species represent an important group of broad-host-range phytopathogens responsible for blackleg and soft rot diseases on numerous plants including many economically important plants. Although these species are commonly detected using cultural, serological, and molecular methods, these methods are sometimes insufficient to classify the bacteria correctly. On that account, this study was undertaken to investigate the feasibility of three individual analytical techniques, capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), for reliable classification of Dickeya and Pectobacterium species. Forty-three strains, representing different Dickeya and Pectobacterium species, namely Dickeya dianthicola, Dickeya dadantii, Dickeya dieffenbachiae, Dickeya chrysanthemi, Dickeya zeae, Dickeya paradisiaca, Dickeya solani, Pectobacterium carotovorum, and Pectobacterium atrosepticum, were selected for this purpose. Furthermore, the selected bacteria included one strain which could not be classified using traditional microbiological methods. Characterization of the bacteria was based on different pI values (CIEF), migration velocities (CZE), or specific mass fingerprints (MALDI-TOF MS) of intact cells. All the examined strains, including the undetermined bacterium, were characterized and classified correctly into respective species. MALDI-TOF MS provided the most reliable results in this respect.

• MeSH
• Electrophoresis, Capillary methods   MeSH
• Enterobacteriaceae chemistry   classification   isolation & purification   MeSH
• Pectobacterium chemistry   classification   isolation & purification   MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Hmotnostní spektrometrie je neodmyslitelnou součástí moderní proteomiky. Při analýzách proteinů, ale i jiných biochemických molekul, nacházejí uplatnění různé typy hmotnostních spektrometrů. Článek se zabývá porovnáním hmotnostních spektrometrů na principu MALDI-TOF (Matrix Assisted Laser Desorption Ionisation–Time of Flight) a LC-Q LC-Q-TOF (Liquid Chromatography Quadrupole Time of Flight) z různých hledisek.

Mass spectrometry is inseparable part of modern proteomics. Different types of mass spectrometers are involved in analysis of proteins or the other biomolecules. The article compares mass spectrometers based on principles MALDI-TOF (Matrix Assisted Laser Desorption Ionisation–Time of Flight) and LC-Q LC-Q-TOF (Liquid Chromatography Quadrupole Time of Flight) from different point of views.

• MeSH
• Chemistry Techniques, Analytical MeSH
• Chromatography, Micellar Electrokinetic Capillary * methods   instrumentation   utilization   MeSH
• Analytic Sample Preparation Methods MeSH
• Intercellular Signaling Peptides and Proteins analysis   classification   MeSH
• Microbiological Techniques MeSH
• Proteins analysis   classification   ultrastructure   MeSH
• Proteomics * methods   instrumentation   MeSH
• Software MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization * methods   instrumentation   utilization   MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH