Comparison of standard and on-plate extraction protocols for identification of mastitis-causing bacteria by MALDI-TOF MS.

The objective was to compare standard versus on-plate sample preparation protocols for identification of mastitis bacteria by MALDI-TOF MS. A total of 186 bacterial isolates from cows with subclinical mastitis were identified by MALDI-TOF MS after preparation using two extraction protocols. On-plate protocol was performed by applying the bacterial colony directly from the culture plate onto the plate spot. For the standard protocol, lysis of bacterial colonies using reagents was performed in a cryotube, and the resulting extract was applied onto the plate spot for analysis. The on-plate protocol showed a similar bacteria identification rate (91.4%, n = 170/186) in comparison to the standard (94.6%, n = 176/186). Identification was higher for both protocols when scores used for species-level identification (≥ 2.0) was reduced to genus-level (≥ 1.7); genus-level identification score rate increased from 94.6 to 100% when using the standard protocol, and from 91.4 to 94.6% when using the on-plate protocol. However, when compared standard (as gold standard) versus on-plate protocol, genus-level identification score rate ranged from 87.1 to 89.8%. Therefore, when the on-plate protocol fails to identify any specie, the standard extraction may be more suitable as a reference protocol for use. Strategy for increasing identification with the on-plate protocol may include upgrading the reference database library. Choice of protocol for preparation may be influenced by the bacterial type to be identified. Standard and on-plate extraction protocols of bacterial ribosomal proteins associated with MALDI-TOF MS might be alternatives to conventional microbiology methods for identification of subclinical mastitis pathogens.

Evaluation of Vitek MS for Differentiation of Cryptococcus neoformans and Cryptococcus gattii Genotypes.

Cryptococcus neoformans and Cryptococcus gattii are the main pathogenic species of invasive cryptococcosis among the Cryptococcus species. Taxonomic studies have shown that these two taxa have different genotypes or molecular types with biological and ecoepidemiological peculiarities. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been proposed as an alternative method for labor-intensive methods for C. neoformans and C. gattii genotype differentiation. However, Vitek MS, one of the commercial MALDI-TOF MS instruments, has not been yet been evaluated for this purpose. Thus, we constructed an in-house database with reference strains belonging to the different C. neoformans (VNI, VNII, VNIII, and VNIV) and C. gattii (VGI, VGII, VGIII, and VGIV) major molecular types by using the software Saramis Premium (bioMérieux, Marcy-l'Etoile, France). Then, this new database was evaluated for discrimination of the different genotypes. Our in-house database provided correct identification for all C. neoformans and C. gattii genotypes; however, due to the intergenotypic mass spectral similarities, a careful postanalytic evaluation is necessary to provide correct genotype identification.

Some coagulase negative Staphylococcus spp. isolated from buffalo can be misidentified as Staphylococcus aureus by phenotypic and Sa442 PCR methods.

OBJECTIVE: Staphylococcus aureus is a commonly reported cause of buffalo mastitis. However, its prevalence may be overestimated. The aim of this study was to compare S. aureus identification by conventional phenotypic and genotypic assays versus Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and novel real-time quantitative PCR tests for the cytochrome oxidase subunit D II (cydB) and staphylocoagulase (coa) genes. RESULTS: From 408 samples obtained from buffalo milk/milking environment, 32 putative S. aureus strains were identified based on characteristic growth on Baird Parker agar, positive catalase reaction, ability to clot rabbit plasma, and positive Sa442 PCR assay. However, in further testing, only 10 of these strains were positive in latex agglutination tests and by MALDI-TOF MS, only eight of the 32 strains were S. aureus while the rest were S. chromogenes (19), S. agnetis (3), S. cohnii (1), or S. xylosus (1). All eight strains identified as S. aureus by MALDI-TOF analysis and confirmed by 16S RNA gene sequencing were positive in a S. aureus-specific cydB PCR test. As well, 7/8 S. aureus strains were PCR positive in a real-time coa PCR test as were 2/69 S. chromogenes and the lone S. xylosus strain tested.

UHPLC-MS and MALDI-MS study of aluminum phthalocyanine chloride and development of a bioanalytical method for its quantification in nanoemulsions and biological matrices.

Metal phthalocyanines are promising components in photodynamic therapy. Aluminum phthalocyanine chloride (AlClPc) has been used to treat oral cancer in mice, human carious tissue, lung cancer cells and other conditions. To overcome the high hydrophobicity of AlClPc, phthalocyanine is often encapsulated in nanoformulations. Despite increased usage, little is known about the pharmacokinetics and biodistribution of AlClPc. The aim of this study was the development and validation of a UHPLC-MS method for the determination of AlClPc in solution after extraction from nanoformulations and biological matrices such as plasma and tissue. The described method has been assayed as to selectivity, linearity, limits of detection and quantification, precision and recovery. The present study is the first to describe the behavior of AlClPc in biological matrices with mass spectrometry as well as the first to describe the chromatographic behavior of AlClPc contaminants. Molecular mass analysis identified dechlorination of AlClPc by both LC/MS and MALDI-MS and an adduct formation in LC/MS. The parameters observed indicated that the method has applicability and robustness for use in biodistribution studies.

MALDI-TOF mass spectrometry applied to identifying species of insect-pathogenic fungi from the Metarhizium anisopliae complex.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a powerful tool for taxonomic resolution of microorganisms. In this proof-of-concept study, we assessed the effectiveness of this technique to track the current gene sequence-based phylogenetic classification of species in the Metarhizium anisopliae complex. Initially the phylogenetic analysis of 5' strains by sequencing of the 59' end of the TEF-1α gene region revealed seven species within M. anisopliae sensu lato and two varieties outside this complex. Because initial studies on MS profiles from different cell types showed that mycelial fragments or conidia produced on nutrient-poor medium may yield too much background noise, all subsequent spectrometric analyses were performed with acidhydrolyzed conidia from 10-12 d old PDA cultures. The initial MALDI-TOF reference library included protein spectral profiles from nine taxonomically distinct, molecularly identified isolates sharing high genetic homology with the ex-type or ex-epitype isolates of these taxa in Metarhizium. A second reference library added one isolate each for M. anisopliae sensu stricto and M. robertsii. The second, larger reference library (including 11 taxa) allowed nearly perfect MALDI-TOF matching of DNA-based species identification for the 40 remaining isolates molecularly recognized as M. anisopliae sensu stricto (n = 19), M. robertsii (n = 6), M. majus (n = 3), M. lepidiotae (n = 1), M. acridum (n = 3), M. flavoviride var. pemphigi (n = 1), plus seven unidentified strains (six of them phylogenetically close to M. anisopliae sensu stricto and one outside the Metarhizium pingshaense-anisopliae-robertsii-brunneum clade). Due to the increasing frequency of phylogenetically (genomically) based taxonomic revisions of fungi, this approach is especially useful for culture collections, because once the protein profiles of Metarhizium isolates are obtained taxonomic updating of MALDI-TOF library data is easily accomplished by comparing stored profiles with those of newly proposed taxa.

Matrix-assisted ultraviolet laser-desorption ionization time-of-flight mass spectrometry of sulfated mannans from the red seaweed Nothogenia fastigiata.

Matrix-assisted ultraviolet laser-desorption ionization time-of-flight mass spectrometry (UV-MALDI-TOF-MS) was applied to sulfated xylo-mannan fractions from Nothogenia fastigiata in order to determine their molecular weights and distribution profiles. The number-average molecular weight calculated from the spectra was similar to that determined by chemical end-group analysis for the lower molecular weight fractions. For the other fractions, the number-average molecular weight was lower than that chemically determined; the increased difference may be attributed to higher desorption difficulties and, consequently, mass-dependent discrimination. A reconstructed spectrum, using the peaks obtained from all the fractions, suggested an unimodal distribution. The best results were obtained by using 2,5-dihydroxybenzoic acid as matrix doped with 1-hydroxyisoquinoline and with harmane and nor-harmane.