Dermatofyty jsou mikroskopické vláknité houby způsobující časté infekce kůže a kožních adnex. Jejich identifikace byla do nedávné doby prováděna pouze podle morfologických charakteristik. Ta je však velmi zdlouhavá a vyžaduje značné zkušenosti. V posledních letech se v identifikaci dermatofytů začíná uplatňovat MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) hmotnostní spektrometrie (MS). U vláknitých hub je nutné, kvůli jejich komplexní morfologii a požadavkům na kultivaci, použít odlišné postupy zpracování kultury. Tato práce se zabývá srovnáním MALDI-TOF MS v identifikaci dermatofytů v porovnání s metodou sekvenace oblasti ITS (Internal Transcribed Spacer) rDNA a konvenčními metodami. Do studie bylo zahrnuto 81 izolátů dermatofytů získaných z klinických vzorků. Výsledky MALDI-TOF MS analýzy se shodovaly s výsledky molekulárně genetické identifikace u 75 % izolátů. Metodami klasické identifikace se podařilo identifikovat pouze 47 % izolátů. MALDI-TOF MS lze tedy považovat za účinný nástroj rychlé a jednoduché identifikace dermatofytů v rutinní diagnostické laboratoři.
Dermatophytes are microscopic fungi causing skin infections and infections of skin adnexa. Their identification is usually performed by morphological characteristics requiring experienced microbiologists and quiet long turnaround time. In recent years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been introducing to many diagnostic laboratories allowing identification of dermatophytes as well. For that purpose, however, specific procedures comparing with bacteria and yeasts must be used because of their complex morphology and specific cultivation requirements. This study is focused on comparison of MALDI-TOF MS-based identification, identification using DNA sequencing of Internal Transcribed Spacer (ITS) rDNA region and conventional methods. In the study, 81 clinical isolates of dermatophytes were included. MALDI-TOF MS correctly identified 74% of all isolates. Using classical methods, only 47% of isolates were identified. Thus, MALDI-TOF MS provides a useful tool for identification of those fungi in routine microbiological laboratories.
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been successfully applied as an identification procedure in clinical microbiology and has been widely used in routine laboratory practice because of its economical and diagnostic benefits. The range of applications of MALDI-TOF MS has been growing constantly, from rapid species identification to labor-intensive proteomic studies of bacterial physiology. The purpose of this review is to summarize the contribution of the studies already performed with MALDI-TOF MS concerning antibiotic resistance and to analyze future perspectives in this field. We believe that current research should continue in four main directions, including the detection of antibiotic modifications by degrading enzymes, the detection of resistance mechanism determinants through proteomic studies of multiresistant bacteria, and the analysis of modifications of target sites, such as ribosomal methylation. The quantification of antibiotics is suggested as a new approach to study influx and efflux in bacterial cells. The results of the presented studies demonstrate that MALDI-TOF MS is a relevant tool for the detection of antibiotic resistance and opens new avenues for both clinical and experimental microbiology.
- MeSH
- antibakteriální látky farmakologie MeSH
- Bacteria účinky léků MeSH
- bakteriální léková rezistence * MeSH
- biomedicínský výzkum trendy MeSH
- mikrobiální testy citlivosti metody MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Nalezení spolehlivé metody detekce karbapenemáz u enterobakterií a některých gramnegativních nefermentujících tyček je velkou výzvou současné klinicko-mikrobiologické diagnostiky. Tento článek shrnuje možnosti detekce karbapenemáz u enterobakterií inhibičními fenotypovými testy, pomocí MALDI-TOF hmotnostní spektrometrie a molekulárně-mikrobiologickými metodami.
Finding a reliable method for the detection of carbapenemases in enterobacteria and some Gram-negative nonfermenting rods is a big challenge for current clinical microbiological diagnosis. This article summarizes the possibilities for carbapenemase detection in enterobacteria provided by inhibitor-based phenotypic tests, MALDI- TOF mass spectrometry, and molecular microbiology techniques.
- Klíčová slova
- KPC, MBL, metropenem, DDST,
- MeSH
- beta-laktamasy analýza izolace a purifikace MeSH
- Enterobacteriaceae izolace a purifikace patogenita MeSH
- fenotyp MeSH
- financování organizované MeSH
- karbapenemy terapeutické užití MeSH
- lidé MeSH
- mikrobiologické techniky metody využití MeSH
- molekulární biologie metody MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody využití MeSH
- Check Tag
- lidé MeSH
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a potentially useful tool for the detection of antimicrobial resistance, especially that conferred by β-lactamases. Here we describe a modification of a previously reported MALDI-TOF MS meropenem hydrolysis assay. The modified method was validated on 108 carbapenemase-producing members of the Enterobacteriaceae, two NDM-1-producing Acinetobacter baumannii isolates, and 35 carbapenem-resistant enterobacteria producing no carbapenemase. The detection of carbapenemases by MALDI-TOF MS seems to be a powerful, quick, and cost-effective method for microbiological laboratories.
- MeSH
- Acinetobacter baumannii enzymologie MeSH
- bakteriální proteiny metabolismus MeSH
- beta-laktamasy metabolismus MeSH
- Enterobacteriaceae enzymologie MeSH
- enterobakteriální infekce mikrobiologie MeSH
- hydrolýza MeSH
- lidé MeSH
- mikrobiální testy citlivosti metody MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- thienamyciny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- validační studie MeSH
Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is used for the determination of molecular weights of different chemical compounds. We describe here the use of MALDI-TOF mass spectrometry to detect a carbapenem antibiotic, meropenem, and its degradation products. Buffered meropenem solution (0.1 mM Tris-HCl, pH 6.8) was mixed with an overnight culture of bacteria. After 3-h incubation, the reaction mixture was centrifuged, and the supernatant was analyzed by MALDI-TOF mass spectrometry. The presence or absence of peaks representing meropenem and its sodium salts was crucial. The average turnaround time of this test, considering the use of overnight culture, is 4 h. We validated this method for the detection of resistance to carbapenems in Enterobacteriaceae and Pseudomonas aeruginosa mediated by carbapenemase production. A total of 124 strains, including 30 carbapenemase-producing strains, were used in the study. The sensitivity of this method is 96.67%, with a specificity of 97.87%. Our results demonstrate the ability of this method to routinely detect carbapenemases in Enterobacteriaceae and Pseudomonas spp. in laboratories. This assay is comparable with a labor-intensive imipenem-hydrolyzing spectrophotometric assay that is a reference method for the detection of carbapenemase. As demonstrated here, MALDI-TOF mass spectrometry may be used in microbiological laboratories not only for microbial identification but also for other applications, such as studies of mechanisms of antibiotic resistance.
- MeSH
- antibakteriální látky analýza MeSH
- bakteriální proteiny metabolismus MeSH
- beta-laktamasy metabolismus MeSH
- Enterobacteriaceae enzymologie MeSH
- lidé MeSH
- Pseudomonas aeruginosa enzymologie MeSH
- senzitivita a specificita MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- thienamyciny analýza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
