MALDI-TOF-MS
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In 2019, Pantoea piersonii was initially isolated from the interior surfaces of the International Space Station. This microorganism is a species within the genus Pantoea in the family Erwiniaceae, belonging to the order Enterobacterales. Recent literature has documented four cases of its isolation. Despite initial predictions suggesting the non-pathogenicity of P. piersonii strains, evidence from observed cases indicates potential pathogenicity. According to documented evidence in the literature, this microorganism is capable of causing severe and life-threatening conditions, including sepsis. Traditional tests, as well as automated systems, may fail to provide complete differentiation due to these similarities. While MALDI-TOF MS is a valuable tool for identification in clinical diagnostic microbiology, sequencing may be necessary for precise identification. To determine the antibiotic susceptibility profile, various methods can be utilized, including minimum inhibitory concentration determination, disk diffusion testing (Kirby-Bauer test), genotypic resistance assays (PCR and sequencing), and automated systems. The literature reports a limited number of cases associating P. piersonii with human infection. This study contributes to this body of knowledge by reporting a novel case in which P. piersonii was isolated from a tissue sample for the first time. In this case report, the patient achieved recovery following the administration of appropriate antibiotic treatment based on the diagnosis. It underscores the need for precise identification and understanding of its pathogenicity.
- MeSH
- antibakteriální látky * farmakologie terapeutické užití MeSH
- enterobakteriální infekce * mikrobiologie diagnóza farmakoterapie MeSH
- lidé MeSH
- mikrobiální testy citlivosti * MeSH
- Pantoea * izolace a purifikace genetika patogenita MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
Corynebacterium (C.) durum je součástí rezidentní flóry dutiny ústní. Jeho podíl na etiologii infekčních onemocnění je nejednoznačný. S vyšším počtem imunoalterovaných pacientů je nutné s ním počítat jako s potenciálním oportunním patogenem. Nejčastěji je izolováno ze sputa, bronchoalveolární lavážní tekutiny, ale také z krve, zejména u imunosuprimovaných pacientů s pneumonií. V tom případě je nutné bakterii přesně identifikovat a nález správně interpretovat. Dříve velmi využívaný komerční test pro určení korynebakterií (API Coryne, BioMerieux) nelze použít pro všechna korynebakteria včetně C. durum. Tento druh není obsažen v databázi biotypových čísel. Lze provést porovnání biotypového čísla s údaji v literatuře. K přesnému odlišení od jiných korynebakterií je nutná chemotaxonomická a proteomická analýzy (MALDI-TOF MS), nebo sekvenace genu 16S rRNA. Klíčový je polyfázový přístup využívající poznatky z jednotlivých laboratorních vyšetření.
Corynebaterium (C.) durum is a part of the resident human oral microbiota. Its role in the aetiology of infectious diseases is ambiguous. With the increasing number of immunocompromised patients, it must be considered a potential opportunistic pathogen. It is isolated from the sputum, bronchoalveolar-lavage fluid, as well as blood, especially from immunocompromised patients with pneumonia. In that case, the critical steps involve a correct identification of Corynebacterium to the species level and right interpretation of the findings. The previously widely used commercial test for the identification of Corynebacteria (API Coryne, BioMerieux) is not suitable for all species, including C. durum, as its biotype number is not included in the database. But the obtained result can be compared with the available literature data. Chemotaxonomic and proteomic analysis (matrix-assisted laser desorption/ ionization – time of flight, MALDI-TOF MS) or 16S rRNA sequencing allow for accurate differentiation from the other Corynebacteria species. Nevertheless, these methods are not routinely used in clinical laboratories. A polyphasic approach to the taxonomy based on the data from combined laboratory tests is crucial.
- MeSH
- bronchoalveolární lavážní tekutina mikrobiologie MeSH
- Corynebacterium * izolace a purifikace MeSH
- imunokompromitovaný pacient MeSH
- korynebakteriální infekce diagnóza MeSH
- krev mikrobiologie MeSH
- lidé MeSH
- mikrobiologické techniky metody MeSH
- oportunní infekce mikrobiologie MeSH
- RNA ribozomální 16S genetika MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- sputum mikrobiologie MeSH
- Check Tag
- lidé MeSH
One of the main challenges in analyzing chemical messengers in the brain is the optimization of tissue sampling and preparation protocols. Limiting postmortem time and terminating enzyme activity is critical to identify low-abundance neurotransmitters and neuropeptides. Here, we used a rapid and uniform conductive heat transfer stabilization method that was compared with a conventional fresh freezing protocol. Together with a selective chemical derivatization method and an optimized quantitation approach using deuterated internal standards, we spatially mapped neurotransmitters and their related metabolites by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) in rat brain tissue sections. Although the heat stabilization did not show differences in the levels of dopamine, norepinephrine, and serotonin, their related metabolites 3,4-dihydroxyphenylacetaldehyde, 3,4-dihydroxyphenylacetic acid, homovanillic acid, 3-methoxy-4-hydroxyphenylacetaldehyde, dihydroxyphenylethyleneglycol, and 5-hydroxyindoleacetic acid were all significantly lower, indicating reduced neurotransmitter postmortem turnover ratios. Heat stabilization enabled detection of an increased number and higher levels of prodynorphin, proenkephalin, and tachykinin-derived bioactive neuropeptides. The low-abundant C-terminal flanking peptide, neuropeptide-γ, and nociceptin remained intact and were exclusively imaged in heat-stabilized brains. Without heat stabilization, degradation fragments of full-length peptides occurred in the fresh frozen tissues. The sample preparation protocols were furthermore tested on rat brains affected by acute anesthesia induced by isoflurane and medetomidine, showing comparable results to non-anesthetized animals on the neurotransmitters level without significant changes. Our data provide evidence for the potential use of heat stabilization prior to MALDI-MSI analyses to improve the examination of the in vivo state of neuronal chemical messengers in brain tissues not impacted by prior acute anesthesia.
- MeSH
- krysa rodu rattus MeSH
- mozek - chemie * fyziologie MeSH
- mozek * metabolismus MeSH
- neurony * metabolismus chemie MeSH
- neurotransmiterové látky * metabolismus analýza MeSH
- potkani Sprague-Dawley MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice * metody MeSH
- vysoká teplota * MeSH
- zmrazování MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Breast milk, as the optimal food for infants and young children, contains all the components necessary for proper growth and development. It is a rich source of both essential nutrients and biologically active factors, making breast milk a unique food with scientifically proven health-promoting properties. Among the entire range of biologically active factors, breast milk microorganisms and prebiotic factors, in the form of breast milk oligosaccharides, occupy an important place. The aim of our research was to determine the occurrence of bacteria with probiotic potential, belonging to the Lactobacillaceae family, in the environment of breast milk and breast milk oligosaccharides. The study included 63 human milk samples from breastfeeding women at various stages of lactation. Microorganism identification based on culture tests and MALDI TOF/MS, macronutrient analysis using the MIRIS human milk analyser, as well as analysis of human milk oligosaccharides using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry were performed. The results have shown that breast milk from different breastfeeding women is characterized by great diversity in terms of the presence of Lacto-bacillaceae bacteria in its microbiological composition. These bacteria were present in 22.2 % of the tested breast milk samples. Analysis of the human milk oligosaccharide profile revealed a slightly higher content of prebiotic factors in breast milk samples containing Lactobacillaceae, including 2'-fucosyllactose, oligosaccharide occurring in the highest amount in breast milk.
Today, MALDI-ToF MS is an established technique to characterize and identify pathogenic bacteria. The technique is increasingly applied by clinical microbiological laboratories that use commercially available complete solutions, including spectra databases covering clinically relevant bacteria. Such databases are validated for clinical, or research applications, but are often less comprehensive concerning highly pathogenic bacteria (HPB). To improve MALDI-ToF MS diagnostics of HPB we initiated a program to develop protocols for reliable and MALDI-compatible microbial inactivation and to acquire mass spectra thereof many years ago. As a result of this project, databases covering HPB, closely related bacteria, and bacteria of clinical relevance have been made publicly available on platforms such as ZENODO. This publication in detail describes the most recent version of this database. The dataset contains a total of 11,055 spectra from altogether 1,601 microbial strains and 264 species and is primarily intended to improve the diagnosis of HPB. We hope that our MALDI-ToF MS data may also be a valuable resource for developing machine learning-based bacterial identification and classification methods.
Candida haemulonii complex (Candida haemulonii [I], Candida duobushaemulonii [II], and Candida haemulonii var. vulnera [III]) has become relevant in recent times, not so much because of a high incidence in human clinical sample cultures but because of its remarkable antifungal resistance. The objective of this study was to evaluate several methods for the identification of this uncommon species of Candida. Ten isolates of C. haemulonii were identified by biochemical and proteomic methods, and their antifungal susceptibility testing was performed by both commercial and reference methods. MALDI-TOF MS (Vitek MS and Vitek MS PRIME) and Vitek2 correctly identified these genera but API method did not. There was a good correlation between the commercial methods and the reference methods for the AST. In conclusion Vitek MS, Vitek MS PRIME, and Vitek2 systems, but not API32C, are reliable for identification of C. haemulonii complex. Furthermore, MALDI-TOF MS systems could identify to the subspecies level. Commercial methods for antifungal susceptibility testing are valid for the study of this species and confirm amphotericin B and to azole resistance.
Cíl práce: Cílem práce je zhodnotit výskyt methicilin-rezistentních kmenů Staphylococcus aureus (MRSA) ve Fakultní nemocnici Olomouc (FNOL) za období 10 let (2013-2022). Materiál a metody: Data byla získána z laboratorního informačního systému ENVIS LIMS (DS Soft, Česká republika, Olomouc) Ústavu mikrobiologie FNOL za období 1. 1. 2013 – 31. 12. 2022. K identifikaci byly použity standardní mikrobiologické postupy s využitím systému MALDI-TOF MS (Biotyper Microflex, Bruker Daltonics). Citlivost k antibiotikům byla stanovena standardní diluční mikrometodou podle kritérií EUCAST. Všechny kmeny Staphylococcus aureus byly testovány na rezistenci k methicilinu pomocí selektivně diagnostické chromogenní půdy (Colorex/TM/MRSA, TRIOS) a imunochromatografického testu na průkaz PBP2a (PBP2a SA Culture Colony Test, AlereTM ). Pozitivní výsledky byly potvrzeny detekcí genu mecA. U izolátů z roku 2022 byla provedena molekulárně-biologická typizace pro stanovení klonality/příbuznosti kmenů pomocí pulzní gelové elektroforézy (PFGE). Výsledky: Prevalence MRSA ve FNOL nevykazuje rostoucí trend a pohybuje se mezi 3-6 %. Nejvyšší prevalence MRSA byla zachycena u materiálů z krevního řečiště (6 %), dále z dolních cest dýchacích (5 %), sekretů z ran a punktátů (5 %). Z jednotlivých oddělení FNOL byla nejvyšší prevalence MRSA na Oddělení geriatrie a II. interní klinice. Profil antibiotické rezistence u souboru izolátů MRSA byl následující: erytromycin 89 %, klindamycin 86 %, ciprofloxacin 80 %, tetracyklin 18 %, gentamicin 13 %, kotrimoxazol 7 %, tigecyklin 1 %. Rezistence k antibiotikům volby v případě závažné infekce způsobené kmenem MRSA (vankomycin, ceftarolin, linezolid) byla 0-1 %. Genetická analýza dvaceti tří izolátů MRSA z roku 2022 pomocí PFGE odhalila jednu pětici, dvě trojice a dvě dvojice se shodným restrikčním profilem. Závěr: Prevalence MRSA ve FNOL je stále nízká, a proto se lze v iniciální terapii infekcí s pravděpodobnou etiologií Staphylococcus aureus spolehnout na oxacilin, případně kombinované aminopeniciliny (amoxicilin/kys. klavulanová, ampicilin/sulbaktam) nebo cefazolin.
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Spinal cord injury (SCI) often leads to central neuropathic pain, a condition associated with significant morbidity and is challenging in terms of the clinical management. Despite extensive efforts, identifying effective biomarkers for neuropathic pain remains elusive. Here we propose a novel approach combining matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with artificial neural networks (ANNs) to discriminate between mass spectral profiles associated with chronic neuropathic pain induced by SCI in female mice. Functional evaluations revealed persistent chronic neuropathic pain following mild SCI as well as minor locomotor disruptions, confirming the value of collecting serum samples. Mass spectra analysis revealed distinct profiles between chronic SCI and sham controls. On applying ANNs, 100% success was achieved in distinguishing between the two groups through the intensities of m/z peaks. Additionally, the ANNs also successfully discriminated between chronic and acute SCI phases. When reflexive pain response data was integrated with mass spectra, there was no improvement in the classification. These findings offer insights into neuropathic pain pathophysiology and underscore the potential of MALDI-TOF MS coupled with ANNs as a diagnostic tool for chronic neuropathic pain, potentially guiding attempts to discover biomarkers and develop treatments.
- MeSH
- biologické markery krev MeSH
- chronická bolest krev diagnóza etiologie MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- neuralgie * krev diagnóza etiologie MeSH
- neuronové sítě * MeSH
- poranění míchy * komplikace krev MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice * metody MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
INTRODUCTION AND PURPOSE: Mycobacterium (M.) chelonae is responsible for a half of relatively rare nontuberculous mycobacteria (NTM) keratitis. We report a case of M. chelonae keratitis in a woman following sclerocorneal suture extraction after cataract surgery. RESULTS: A 70-year-old woman presented with a red eye and corneal infiltration of her left eye six weeks following sclerocorneal suture extraction after an elective cataract surgery in another institute. She complained of a sharp, cutting pain and photophobia. Since initial corneal scrapes and conjunctival swabs proved no pathogen using culture and PCR methods, non-specific antibiotics and antifungal agents were administered. As keratitis was complicated by an inflammation in the anterior chamber and vitreous, samples of the vitreous fluid were sent for microbiologic examination. DNA of Epstein-Barr virus (EBV) was repeatedly detected. Since the intrastromal abscess had formed, corneal re-scrapings were performed and M. chelonae was detected using culture, MALDI-TOF MS and PCR methods. Therapy was changed to a combination of oral and topical clarithromycin, intravitreal, topical and intracameral amikacin, and oral and topical moxifloxacin. The successful therapy led to stabilization. The optical penetrating keratoplasty was performed and no signs of the infection recurrence were found. CONCLUSIONS: The diagnosis of nontuberculous mycobacterial keratitis is difficult and often delayed. An aggressive and prolonged antimicrobial therapy should include systemic and topical antibiotics. Surgical intervention in the form of corneal transplantation may be required in the active and nonresponsive infection. In the presented case this was necessary for visual rehabilitation due to scarring.
- MeSH
- amikacin terapeutické užití MeSH
- antibakteriální látky * terapeutické užití MeSH
- atypické mykobakteriální infekce * diagnóza farmakoterapie mikrobiologie chirurgie MeSH
- fluorochinolony terapeutické užití MeSH
- keratitida * diagnóza farmakoterapie mikrobiologie chirurgie MeSH
- klarithromycin terapeutické užití MeSH
- lidé MeSH
- moxifloxacin * terapeutické užití MeSH
- Mycobacterium chelonae * izolace a purifikace MeSH
- senioři MeSH
- výsledek terapie MeSH
- Check Tag
- lidé MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- přehledy MeSH
- Geografické názvy
- Evropa MeSH
The MBT Pathfinder is an automated colony-picking robot designed for efficient sample preparation in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This article presents results from three key experiments evaluating the instrument's performance in conjunction with MALDI Biotyper instrument. The method comparison experiment assessed its clinical performance, demonstrating comparable results with gram-positive, gram-negative, and anaerobic bacteria (scores larger than 2.00) and superior performance over simple direct yeast transfer (score: 1.80) when compared to samples prepared manually. The repeatability experiment confirmed consistent performance over multiple days and labs (average log score: 2.12, std. deviation: 0.59). The challenge panel experiment showcased its consistent and accurate performance across various samples and settings, yielding average scores between 1.76 and 2.19. These findings underline the MBT Pathfinder as a reliable and efficient tool for MALDI-TOF mass spectrometry sample preparation in clinical and research applications.
- MeSH
- Bacteria * klasifikace izolace a purifikace MeSH
- laboratorní automatizace * metody MeSH
- lidé MeSH
- odběr biologického vzorku metody MeSH
- reprodukovatelnost výsledků MeSH
- robotika * MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
