Matrix-assisted laser desorption ionization-time of flight mass spectrometry for the discrimination of food-borne microorganisms.
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ABSTRACT:
SUBMITTER: Mazzeo MF
PROVIDER: S-EPMC1392959 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:More than 20 different species of Mucorales can be responsible for human mucormycosis. Accurate identification to the species level is important. The morphological identification of Mucorales is not reliable, and the currently recommended identification standard is the molecular technique of sequencing the internal transcribed spacer regions. Nevertheless, matrix-assisted laser desorption ionization time-of-flight mass spectrometry has been shown to be an accurate alternative for the identification of bacteria, yeasts, and even filamentous fungi. Therefore, 38 Mucorales isolates, belonging to 12 different species or varieties, mainly from international collections, including 10 type or neo-type strains previously identified by molecular methods, were used to evaluate the usefulness of matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the identification of human pathogenic Mucorales to the species level. One to three reference strains for each species were used to create a database of main spectrum profiles, and the remaining isolates were used as test isolates. A minimum of 10 spectra was used to build the main spectrum profile of each database strain. Interspecies discrimination for all the isolates, including species belonging to the same genus, was possible. Twenty isolates belonging to five species were used to test the database accuracy, and were correctly identified to the species level with a log-score >2. In summary, matrix-assisted laser desorption ionization time-of-flight mass spectrometry is a reliable and rapid method for the identification of most of the human pathogenic Mucorales to the species level.
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Project description:Although some Weissella species play beneficial roles in food fermentation and in probiotic products, others such as Weissella confusa are emerging Gram-positive pathogens in immunocompromised hosts. Weissella species are difficult to identify by conventional biochemical methods and commercial automated systems and are easily misidentified as Lactobacillus and Leuconostoc species. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly being used for bacterial identification. Little, however, is known about the effectiveness of MALDI-TOF MS in identifying clinical isolates of Weissella to the species level. In this study, we evaluated whether the MALDI-TOF MS Bruker Biotyper system could accurately identify a total of 20 W. confusa and 2 W. cibaria blood isolates that had been confirmed by 16s rRNA sequencing analysis. The MALDI-TOF Biotyper system yielded no reliable identification results based on the current reference spectra for the two species (all score values <1.7). New W. confusa spectra were created by randomly selecting 3 W. confusa isolates and external validation was performed by testing the remaining 17 W. confusa isolates using the new spectra. The new main spectra projection (MSP) yielded reliable score values of >2 for all isolates with the exception of one (score value, 1.963). Our results showed that the MSPs in the current database are not sufficient for correctly identifying W. confusa or W. cibaria. Further studies including more Weissella isolates are warranted to further validate the performance of MALDI-TOF in identifying Weissella species.
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Project description:Antibiotic resistance in Gram-negative microorganisms is an increasing health care problem. The rapid detection of such resistance is crucial for starting an early specific therapy and to enable initiation of the required hygiene measures. With continued emphasis on reducing the cost of laboratory testing, only economical/low-cost approaches have a chance of being implemented. During recent years, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been developed to be a standard method in microbiology laboratories for the rapid and cost-efficient identification of microorganisms. Extending the usage of MALDI-TOF MS in the clinical microbiology laboratory to the area of resistance testing is an attractive option. Quantitative MALDI-TOF MS using an internal standard facilitates the measurement of the quantity of peptides and small proteins within a spectrum. These quantities correlate to the number of microorganisms and therefore to the growth of a microorganism. The comparison of growth in the presence or absence of an antibiotic allows for analysis of the susceptibility behavior of a strain. Here, we describe a novel method and its application in the analysis of 108 Klebsiella sp. isolates. After 1 h of incubation at a meropenem concentration of 8 ?g/ml, a sensitivity of 97.3% and a specificity of 93.5% were achieved (compared to Etest results).
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Project description:The rapid and cost-efficient determination of carbapenem resistance is an important prerequisite for the choice of an adequate antibiotic therapy. A MALDI-TOF MS-based assay was set up to detect porins in the current study. A loss of the components of porin alone such as OmpK35/OmpK36 or together with the production of carbapenemases will augment the carbapenem resistance. Ten strains of Escherichia coli and eight strains of Klebsiella pneumoniae were conducted for both sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and MALDI-TOF MS analysis. MALDI-TOF/TOF MS analysis was then performed to verify the correspondence of proteins between SDS-PAGE and MALDI-TOF MS. The results indicated that the mass spectrum of ca. 35,000, 37,000, and 38,000-m/z peaks of E. coli ATCC 25922 corresponded to OmpA, OmpC, and OmpF with molecular weight of approximately ca. 38, 40, and 41 kDa in SDS-PAGE gel, respectively. The band of OmpC and OmpF porins were unable to be distinguished by SDS-PAGE, whereas it was easy to be differentiated by MALDI-TOF MS. As for K. pneumoniae isolates, the mass spectrum of ca. 36,000 and 38,600-m/z peaks was observed corresponding to OmpA and OmpK36 with molecular weight of approximately ca. 40 and 42 kDa in SDS-PAGE gel, respectively. Porin OmpK35 was not observed in the current SDS-PAGE, while a 37,000-m/z peak was found in K. pneumoniae ATCC 13883 and carbapenem-susceptible strains by MALDI-TOF MS which was presumed to be the characteristic peak of the OmpK35 porin. Compared with SDS-PAGE, MALDI-TOF MS is able to rapidly identify the porin-deficient strains within half an hour with better sensitivity, less cost, and is easier to operate and has less interference.
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Project description:In this study, Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry was used to identify Mycobacterium bovis from cattle and buffalo tissue isolates from the North and South regions of Brazil, grown in solid medium and previously identified by Polymerase Chain Reaction (PCR) based on Region of Difference 4 (RD4), sequencing and spoligotyping. For this purpose, the protein extraction protocol and the mass spectra reference database were optimized for the identification of 80 clinical isolates of mycobacteria. As a result of this optimization, it was possible to identify and differentiate M. bovis from other members of the Mycobacterium tuberculosis complex with 100% specificity, 90.91% sensitivity and 91.25% reliability. MALDI-TOF MS methodology described herein provides successful identification of M. bovis within bovine/bubaline clinical samples, demonstrating its usefulness for bovine tuberculosis diagnosis in the future.
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Project description:The widespread use of antifungal agents, which is likely to expand with their enhanced availability, has promoted the emergence of drug-resistant strains. Antifungal susceptibility testing (AFST) is now an essential procedure for guiding appropriate antifungal therapy. Recently, we developed a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based method that enables the detection of fungal isolates with reduced echinocandin susceptibility, relying on the proteome changes that are detectable after a 15-h exposure of fungal cells to serial drug concentrations. Here, we describe a simplified version of this approach that facilitates discrimination of the susceptible and resistant isolates of Candida albicans after a 3-h incubation in the presence of "breakpoint" level drug concentrations of the echinocandin caspofungin (CSF). Spectra at concentrations of 0 (null), 0.03 (intermediate), and 32 (maximal) ?g/ml of CSF were used to create individual composite correlation index (CCI) matrices for 65 C. albicans isolates, including 13 fks1 mutants. Isolates are then classified as susceptible or resistant to CSF if the CCI values of spectra at 0.03 and 32 ?g/ml are higher or lower, respectively, than the CCI values of spectra at 0.03 and 0 ?g/ml. In this way, the drug resistance of C. albicans isolates to echinocandin antifungals can be quickly assessed. Furthermore, the isolate categorizations determined using MALDI-TOF MS-based AFST (ms-AFST) were consistent with the wild-type and mutant FKS1 genotypes and the AFST reference methodology. The ms-AFST approach may provide a rapid and reliable means of detecting emerging antifungal resistance and accelerating the initiation of appropriate antifungal treatment.
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Project description:Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a relatively new addition to the clinical microbiology laboratory. The performance of the MALDI Biotyper system (Bruker Daltonics) was compared to those of phenotypic and genotypic identification methods for 690 routine and referred clinical isolates representing 102 genera and 225 unique species. We systematically compared direct-smear and extraction methods on a taxonomically diverse collection of isolates. The optimal score thresholds for bacterial identification were determined, and an approach to address multiple divergent results above these thresholds was evaluated. Analysis of identification scores revealed optimal species- and genus-level identification thresholds of 1.9 and 1.7, with 91.9% and 97.0% of isolates correctly identified to species and genus levels, respectively. Not surprisingly, routinely encountered isolates showed higher concordance than did uncommon isolates. The extraction method yielded higher scores than the direct-smear method for 78.3% of isolates. Incorrect species were reported in the top 10 results for 19.4% of isolates, and although there was no obvious cutoff to eliminate all of these ambiguities, a 10% score differential between the top match and additional species may be useful to limit the need for additional testing to reach single-species-level identifications.
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