Browsing by Subject "MALDI-TOF MS"

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  • Huotari, Jaana (Helsingin yliopisto, 2020)
    This study aimed to investigate the microbiological quality of the whole and gutted Baltic herring at different seasons by traditional culture-dependent methods combined with the identification of bacterial isolates by MALDI-TOF MS. Additionally, the microbiome of the herring was characterized by culture-independent 16S rRNA gene amplicon sequencing. Bacterial counts were within acceptable limits at all seasons although the H2S-producing bacteria levels were above the recommended level of 5 log10 CFU g-1 at two sampling points. With the culture-dependent methods and the sequencing of the 16S rRNA gene, the microbiome of the whole and gutted herring was dominated by the bacterial class Gammaproteobacteria. Shewanella, Pseudomonas, and Aeromonas were the most frequently isolated genera among the viable population identified with MALDI-TOF MS. With the culture-independent approach, Shewanella followed by Psychrobacter were the most abundant genera. Additionally, a high relative abundance of the phyla Firmicutes and Actinobacteria and, in some samples, Epsilonbacteriaeota represented by the genus Arcobacter, was detected. Variances in the microbiological quality of different herring batches observed in this study could not be attributed to the season. Therefore future research through a longer period was proposed, including data on the environmental factors, such as the fishing location and the water quality, possibly affecting the quality of the herring.
  • Smith, Colin (Helsingin yliopisto, 2020)
    The Genome Encyclopedia of Spacecraft Associated Microbes (GESAM) project seeks to increase our knowledge of spacecraft associated microbes surviving in the extreme environments of spacecraft associated clean rooms. Planetary Protection researchers at NASA’s Jet Propulsion Laboratory have been collecting and archiving bacterial isolates from NASA Mars missions since the 1970s, however, only a small fraction of the entire bacterial collection has been taxonomically identified and even fewer have been functionally characterized. Previous efforts to taxonomically identify microbes employed 16S rRNA gene sequencing techniques, however, this method alone failed to provide the resolution required to consistently identify these isolates at a species and strain level, many of which are members of taxonomic groups further challenging identification. Samples collected from spacecraft surfaces and within cleanrooms throughout assembly testing and launch operations were subjected to NASA’s heat shock assay and stored for future analysis. In this thesis, for the first time, Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) has been used to identify bacterial strains from the Mars Odyssey and Mars Pathfinder missions. Due to its clinically biased design framework, the development of an in-house database of mass spectrum profiles complemented with 16S rRNA gene sequencing-based taxonomic IDs was necessary to identify this project’s isolates which reside in extreme environments of spacecraft assembly cleanrooms and are not well represented in existing shared spectral databases. MALDI-TOF MS was found to be a fast, reliable, and powerful method of identifying bacterial isolates at species and strain level. Despite MALDI-TOF’s high resolving power, its ability to identify space associated microbes residing in spacecraft assembly cleanrooms is hindered by the reliance of mass spectrum profile creation on 16S rRNA gene taxonomic identification, and the lack of shared spectrum data and publicly available databases. The resolution of taxonomic groups via composite correlation index analysis is paramount to the development of spectra libraries and the utilization of MALDI to its full potential. While MALDI is well suited to identifying the proxy representation of cultivatable spore-forming bacteria produced by NASA’s Standard Assay, a surprising number of non-spore-forming have been identified and many more are expected to be uncultivatable suggesting the need for less selective omics-based approaches in order to establish a more comprehensive idea of the communities present in spacecraft assembly cleanroom.
  • Aarnio, Sanna (Helsingfors universitet, 2017)
    Matriisiavusteinen massaspektrometria (Matrix Assisted Laser Desorption/Ionization Time Of Flight Mass Spectrometry, MALDI-TOF MS) on molekyylien massa-varaussuhteen mittaamiseen perustuva analyysimenetelmä. MALDI-TOF-massaspektrometriaa käytetään mikrobien lajintunnistukseen, mutta joillain mikrobilajeilla se saattaa soveltua myös lajitasoa tarkempaan analytiikkaan. MALDI-TOF-massaspektrometrian käytöstä Staphylococcus aureuksen metisilliiniresistenssiominaisuuden tunnistuksessa ja epi- demiologisessa tyypityksessä on saatu lupaavia tuloksia. Staphylococcus pseudintermediuksella vastaavaa tutkimusta ei ole aiemmin tehty. Tässä työssä selvitettiin MALDI-TOF-massaspektrometrian soveltuvuutta S. pseudintermedius -kantojen metisilliini-resistenssiominaisuuden tunnistukseen ja MRSP (metisilliinille resistenttien S. pseudintermedius) -kantojen epidemiologiseen tyypitykseen. Metisilliiniresistenssiominaisuuden tunnistuksessa tutkittiin 10 MRSP- kantaa ja 10 MSSP (metisilliinille sensitiivistä S. pseudinter-medius) -kantaa. MRSP:n epidemiologisessa tyypityksessä tutkittiin 15 MRSP-kantaa, joista kutakin tutkittua klonaalista kompleksia (CC45, CC71 ja CC258) edusti viisi kantaa. Massaspektrit mitattiin Bruker Microflex LT 3.1 -laitteella, ja spektrit käsiteltiin Flex-Analysis 3.4 -ohjelmalla. Erottavia huippuja etsittiin visuaaliseen spektritarkasteluun perustuvan kantakohtaisen huipputaulukoinnin avulla. MRSP-kannoista etsittiin paitsi erottavia huippuja yleisesti, myös kohdennetusti metisilliiniresistenssiominaisuuteen liittyvien PSM-mec- ja MecI-proteiinien massaa vastaavia huippuja. Visuaalisen analyysin lisäksi spektreistä luotiin kantakohtaiset kokoomaspektrit (Main Spectral Profile, MSP), joiden samankaltaisuutta tarkasteltiin FlexAnalysis-ohjelman klusterianalyysillä. MRSP- ja MSSP- kantojen spektrejä ei pystytty erottamaan toisistaan. MecI-proteiinin massaa vastaavia huippuja havaittiin sekä MRSP- että MSSP-kannoilla, ja PSM-mec-proteiinin massaa vastaava huippu havaittiin yhden MRSP-kannan isolaatilla. Klusterianalyysissä MRSP-kannat ryvästyivät sekaisin MSSP-kantojen kanssa. Kaikki klonaaliset MRSP-kompleksit pystyttiin erottamaan toisistaan huippujen 2047 +/-2 m/z ja 4290 +/-3 m/z esiintymisen perusteella. 2047 +/-2 m/z esiintyi kaikilla klonaalisen kompleksin CC258 kannoilla, mutta ei millään muilla kannoilla. 4290 +/-3 m/z esiintyi kaikilla CC71-kannoilla, mutta ei CC45-kannoilla. Eroja havaittiin myös useiden muiden huippujen esiintymisessä. Klusterianalyysissä klonaaliset kompleksit eivät erottuneet toisistaan. Tämän tutkimuksen perusteella MALDI-TOF saattaa soveltua joidenkin klonaalisten MRSP-kompleksien erotteluun, mutta menetelmän käytölle S. pseudintermediuksen metisilliiniresistenssin tunnistamisessa ei saatu puoltavaa näyttöä. Klonaalisten kompleksien erottelussa huippujen poiminta saattaa toimia paremmin kuin klusterianalyysi.
  • Hallamaa, Raija; Batchu, Krishna (2016)
    Background: Lipids have become an important target for searching new biomarkers typical of different autoimmune and allergic diseases. The most common allergic dermatitis of the horse is related to stings of insects and is known as insect bite hypersensitivity (IBH) or summer eczema, referring to its recurrence during the summer months. This intense pruritus has certain similarities with atopic dermatitis of humans. The treatment of IBH is difficult and therefore new strategies for therapy are needed. Autoserum therapy based on the use of serum phospholipids has recently been introduced for horses. So far, serum lipids relating to these allergic disorders have been poorly determined. The main aim of this study was to analyse phospholipid profiles in the sera of horses with allergic dermatitis and in their healthy controls and to further assess whether these lipid profiles change according to the clinical status after therapy. Methods: Sera were collected from 10 horses with allergic dermatitis and from 10 matched healthy controls both before and 4 weeks after the therapy of the affected horses. Eczema horses were treated with an autogenous preparation made from a horse's own serum and used for oral medication. Samples were analysed for their phospholipid content by liquid chromatography coupled to a triple-quadrupole mass spectrometer (LC-MS). Data of phospholipid concentrations between the groups and over the time were analysed by using the Friedman test. Correlations between the change of concentrations and the clinical status were assessed by Spearman's rank correlation test. Results: The major phospholipid classes detected were phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Eczema horses had significantly lower total concentrations of PC (p <0.0001) and SM (p = 0.0115) than their healthy controls. After a 4-week therapy, no significant differences were found between the groups. Changes in SM concentrations correlated significantly with alterations in clinical signs (p = 0.0047). Conclusions: Horses with allergic dermatitis have an altered phospholipid profile in their sera as compared with healthy horses and these profiles seem to change according to their clinical status. Sphingomyelin seems to have an active role in the course of equine insect bite hypersensitivity.