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Echinocandins are the recommended first-line antifungals for treatment of invasive candidiasis. The increasing number of Candida glabrata strains resistant against echinocandins is an emerging health care concern. The rapid detection of resistant C. glabrata isolates is an urgent requirement for clinical laboratories. In this study, we developed the MALDI Biotyper antibiotic (antifungal) susceptibility test rapid assay (MBT ASTRA) for the rapid detection of anidulafungin-resistant C. glabrata isolates directly from positive blood cultures. Of 100  C. glabrata strains, MBT ASTRA classified 69 as susceptible and 29 as resistant. Microdilution assays performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, used as a standard reference, identified 65 susceptible, 9 intermediate, and 26 resistant isolates. Sequencing of hot spot 1 and hot spot 2 regions of the FKS1 and FKS2 genes classified 86 susceptible and 14 resistant isolates. The MBT ASTRA had sensitivity and specificity of 80% and 95%, respectively, compared to the microdilution method. Positive and negative agreement of MBT ASTRA was calculated at 100% and 80%, respectively, compared with the molecular sequencing approach. Together, these results revealed a high accuracy of MBT ASTRA compared to microdilution according to the CLSI and PCR analysis, resulting in a categorical agreement of 90% and 83%, respectively. The validity of MBT ASTRA was 98%. Importantly, MBT ASTRA provided antifungal susceptibility testing (AFST) within 6 h that was both accurate and reliable compared to the other two approaches, which require at least 24 h or are costly. Therefore, this method has the potential to facilitate clinical AFST rapidly at low sample costs for clinical labs already equipped with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)., (Copyright © 2019 American Society for Microbiology.)

The present study gives a detailed phenotypic and genotypic characterization of three Trueperella abortisuis strains isolated from a ten year old male Hovawart dog with an abscess of anal sac, from urine of an eight year old European shorthair cat with urolithiasis and nephrolithiasis and from a 14year old Maine Coon cat with a perianal abscess, respectively. All three strains could be identified phenotypically, by MALDI-TOF MS analysis and genotypically by sequencing the 16S rDNA and the molecular target genes gap and tuf. The present study gives a first description of T. abortisuis of this origin., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Introduction : Ten years after its introduction into clinical microbiology, MALDI-TOF mass spectrometry has become the standard routine identification tool for bacteria in most laboratories. The technology has accelerated analyses and improved the quality of results. The greatest significance has been observed for bacteria that were challenging to be identified by traditional methods. Areas covered : We searched in existing literature (Pubmed) for reports how MALDI-TOF MS has contributed to identification of rare and unknown bacteria from different groups. We describe how this has improved the diagnostics in different groups of bacteria. Reference patterns for strains which yet cannot be assigned to a known species even enable the search for related bacteria in studies as well as in routine diagnostics. MALDI-TOF MS can help to discover and investigate new species and their clinical relevance. It is a powerful tool in the elucidation of the bacterial composition of complex microbiota in culturomics studies. Expert opinion : MALDI-TOF MS has improved the diagnosis of bacterial infections. It also enables knowledge generation for prospective diagnostics. The term 'hard-to-identify' might only be rarely attributed to bacteria in the future. Novel applications are being developed, e.g. subspecies differentiation, typing, and antibiotic resistance testing which may further contribute to improved microbial diagnostics.

Annotation and identification of metabolite biomarkers is critical for their biological interpretation in metabolic phenotyping studies, presenting a significant bottleneck in the successful implementation of untargeted metabolomics. Here, a systematic multistep protocol was developed for the purification and de novo structural elucidation of urinary metabolites. The protocol is most suited for instances where structure elucidation and metabolite annotation are critical for the downstream biological interpretation of metabolic phenotyping studies. First, a bulk urine pool was desalted using ion-exchange resins enabling large-scale fractionation using precise iterations of analytical scale chromatography. Primary urine fractions were collected and assembled into a "fraction bank" suitable for long-term laboratory storage. Secondary and tertiary fractionations exploited differences in selectivity across a range of reversed-phase chemistries, achieving the purification of metabolites of interest yielding an amount of material suitable for chemical characterization. To exemplify the application of the systematic workflow in a diverse set of cases, four metabolites with a range of physicochemical properties were selected and purified from urine and subjected to chemical formula and structure elucidation by respective magnetic resonance mass spectrometry (MRMS) and NMR analyses. Their structures were fully assigned as tetrahydropentoxyline, indole-3-acetic-acid- O -glucuronide, p -cresol glucuronide, and pregnanediol-3-glucuronide. Unused effluent was collected, dried, and returned to the fraction bank, demonstrating the viability of the system for repeat use in metabolite annotation with a high degree of efficiency.

Establishing an effective empirical antifungal therapy requires that national surveillance studies be conducted. Herein, we report the clinical outcome of infections with and the microbiological features of Iranian isolates of Candida glabrata derived from patients suffering from candidemia. C. glabrata isolates were retrospectively collected from four major cities in Iran; identified by a 21-plex PCR, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and large subunit of ribosomal DNA sequencing; and genotyped by amplified fragment length polymorphism (AFLP). Mutations in PDR1 , ERG11 , and hot spot 1 (HS1) of FKS1 and FKS2 were investigated, and antifungal susceptibility testing (AFST) was performed (by the CLSI M27-A3 and M27-S4 methods). Seventy isolates of C. glabrata were collected from 65 patients with a median age of 58 years. Fluconazole was the most widely used (29.23%) and least effective antifungal agent. The overall crude mortality rate was 35.4%. Only one strain was resistant to fluconazole, and 57.7% and 37.5% of the isolates were non-wild type (non-WT) for susceptibility to caspofungin and voriconazole, respectively. All isolates showed the WT phenotype for amphotericin B, posaconazole, and itraconazole. HS1 of FKS1 and FKS2 did not harbor any mutations, while numerous missense mutations were observed in PDR1 and ERG11 AFLP clustered our isolates into nine genotypes; among them, genotypes 1 and 2 were significantly associated with a higher mortality rate ( P  = 0.034 and P  = 0.022, α < 0.05). Moreover, 83.3% of patients infected with strains harboring a single new mutation in PDR1 , T745A, died despite treatment with fluconazole or caspofungin. Overall, Iranian isolates of C. glabrata were susceptible to the major antifungal drugs. Application of genotyping techniques and sequencing of a specific gene ( PDR1 ) might have prognostic implications., (Copyright © 2019 Arastehfar et al.)

The spread of bacterial resistance has been continuously increasing in the recent decade. Multi-drug resistant (MDR) bacteria now represent one of the most worrisome public health issues, as they seriously complicate the treatment of infections, often leaving few therapeutic options. Enterobacteria and Staphylococcus aureus are among the most common bacterial pathogens, while Bacteroides fragilis is the most frequent anaerobic pathogen. All of these species can cause severe and life-threatening infections, and represent the most frequent causes of antibiotic-resistant healthcare-associated infections worldwide, as they frequently exhibit resistance to various classes of antibiotics. Resistance to carbapenems, the last resort beta-lactam agent, is a particularly threatening problem. Achieved by different mechanisms, leads to total inefficacy of any beta-lactam agent. During the recent years, MALDI-TOF mass spectrometry has become established as the reference method for bacterial identification in routine practice. It has proven to be a reliable and robust method to detect specific peaks in bacterial mass spectra, corresponding to specific resistance markers, enabling the instant detection of resistant isolates in real time during the standard routine identification process. Here, we investigated the performance of the subtyping module of the MALDI Biotyper system (Bruker Daltonik, GmbH) for the instant identification of KPC-producing Klebsiella pneumoniae , methicillin-resistant Staphylococcus aureus , and carbapenemase-producing Bacteroides fragilis during the identification workflow. We evaluated accuracy and potential impact on turnaround time. Furthermore, we investigated the possibility to extend the subtyping for detection of the KPC-specific marker to bacterial species other than K. pneumoniae ., Competing Interests: MC, ABP and SA have any conflicts of interest to disclose. Authors with financial interests or relationships to disclose are listed with their details below: MK is employee of Bruker Daltonik GmbH, manufacturer of the MALDI Biotyper system. Corresponding author confirms here the conflict of interest statements provided during the initial submission of the manuscript., (© 2019 The Association for Mass Spectrometry: Applications to the Clinical Lab (MSACL). Published by Elsevier B.V.)

In the presented work identification of microorganisms isolated from various types of honeys was performed. Martix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rDNA sequencing were applied to study environmental bacteria strains.With both approches, problematic spore-forming Bacillus spp, but also Staphylococcus spp., Lysinibacillus spp., Micrococcus spp. and Brevibacillus spp were identified. However, application of spectrometric technique allows for an unambiguous distinction between species/species groups e.g.B. subtilis or B. cereus groups. MALDI TOF MS and 16S rDNA sequencing allow for construction of phyloproteomic and phylogenetic trees of identified bacterial species. Furthermore, the correlation beetween physicochemical properties, geographical and botanical origin and the presence bacterial species in honey samples were investigated., Competing Interests: The authors have read the journal's policy and the authors of this manuscript have the following competing interests: MK is a paid employee of the Bruker Daltonik GmbH company. Bruker Daltonik GmbH company was not an additional funder of the study nor had influence on the study design or interpretation. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

In this work, uniform manifold approximation and projection (UMAP) is applied for nonlinear dimensionality reduction and visualization of mass spectrometry imaging (MSI) data. We evaluate the performance of the UMAP algorithm on MSI data sets acquired in mouse pancreas and human lymphoma samples and compare it to those of principal component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE), and the Barnes-Hut (BH) approximation of t-SNE. Furthermore, we compare different distance metrics in (BH) t-SNE and UMAP and propose the use of spatial autocorrelation as a means of comparing the resulting low-dimensional embeddings. The results indicate that UMAP is competitive with t-SNE in terms of visualization and is well-suited for the dimensionality reduction of large (>100 000 pixels) MSI data sets. With an almost fourfold decrease in runtime, it is more scalable in comparison with the current state-of-the-art: t-SNE or the Barnes-Hut approximation of t-SNE. In what seems to be the first application of UMAP to MSI data, we assess the value of applying alternative distance metrics, such as the correlation, cosine, and the Chebyshev metric, in contrast to the traditionally used Euclidean distance metric. Furthermore, we propose "histomatch" as an additional custom distance metric for the analysis of MSI data.

Dynamically harmonized FT-ICR cell has a saddle-like hyperbolic field distribution inside when averaged over a cyclotron trajectory around the axis of the cell. Such a field distribution makes the motion along the magnetic field independent of the motion in the x,y-plane, as well as the cyclotron motion independent of the magnetron motion and prevents any disintegration of excited coherent ion clouds, which is ruining the resolution in the other types of FT-ICR cells providing by this ideal phasing of single-m/z ion clouds in the entire volume of the cell. FT-ICR instruments with such a cell show resolutions of more than ten million at m/z 1000 at relatively small magnetic fields like 7 Tesla in quadrupole detection mode, what is not reachable by any other type of modern mass spectrometers. We have found that for such ion traps, it is possible to find the analytical solution in the working volume of the trap without any averaging. The potential distribution for the almost whole volume of such a cell can be presented in the form ϕ(x, y, z) = αz 2  + f 2D (x, y), where f 2D (x, y) is the solution of 2D Poisson equation, which could be found by the method of conformal transformation. This solution is applicable in the practical case and can serve as a base for an analytical theory of signal detection using such cells and as a standard for solutions obtained by numerical simulations of the cell field.

Staten Island is located in one of the most densely populated regions of the US: the New York/New Jersey Estuary. Marine and industrial oil spills are commonplace in the area, causing the waterways and adjacent marshes to become polluted with a range of petroleum-related contaminants. Using Rock-Eval pyrolysis, the hydrocarbon impact on a salt marsh was assessed at regular intervals down to 90 cm, with several key sampling depths of interest identified for further analysis. Ultrahigh resolution data are obtained by direct infusion (DI) atmospheric pressure photoionization (APPI) on a 12 T solariX Fourier transform ion cyclotron resonance mass spectrometer (FTICR MS) allowing trends in the compositional profile with depth to be observed, such as changes in the relative hydrocarbon intensity and the relative contributions from oxygen- and sulfur-containing groups. These trends may correlate with the timing of major oil spills and leaks of petroleum and other industrial chemicals into the waterways. The use of gas chromatography (GC) coupled to a 7 T solariX 2XR FTICR MS equipped with an atmospheric pressure chemical ionization (APCI) ion source offers retention time resolved and extensive compositional information for the complex environmental samples complementary to that obtained by DI-APPI. The compositional profile observed using GC-APCI FTICR MS includes contributions from phosphorous-containing groups, which may be indicative of contamination from other anthropogenic sources., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Methylobacterium radiotolerans is a fastidious, pink-pigmented, obligate aerobic Gram-negative bacillus commonly isolated from various environmental sources, and only occasionally from clinical samples, mostly in immunocompromised patients or associated with intravascular devices and haemodialysis. It grows poorly on commonly used culture media and its identification is time-consuming by conventional means. In this study, we present a case of M. radiotolerans bacteraemia in an individual affected by end-stage renal failure, identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The species identification was further confirmed by biochemical and molecular methods. The susceptibility to various antimicrobial agents is also presented and discussed.

Comparative 16S rRNA gene sequence analysis and major physiological differences indicate two distinct sublineages within the genus Azoarcus: the Azoarcus evansii lineage, comprising Azoarcusevansii (type strain KB740 T =DSM 6898 T =CIP 109473 T =NBRC 107771 T ), Azoarcusbuckelii (type strain U120 T =DSM 14744 T =LMG 26916 T ), Azoarcusanaerobius (type strain LuFRes1 T =DSM 12081 T =LMG 30943 T ), Azoarcustolulyticus (type strain Tol-4 T =ATCC 51758 T =CIP 109470 T ), Azoarcustoluvorans (type strain Td21 T =ATCC 700604 T =DSM 15124 T ) and Azoarcustoluclasticus (type strain MF63 T =ATCC 700605 T ), and the Azoarcus indigens lineage, comprising Azoarcusindigens (type strain VB32 T =ATCC 51398 T =LMG 9092 T ), Azoarcus communis (type strain SWub3 T =ATCC 51397 T =LMG 9095 T ) and Azoarcusolearius (type strain DQS-4 T =BCRC 80407 T =KCTC 23918 T =LMG 26893 T ). Az. evansii lineage members have remarkable anaerobic degradation capacities encompassing a multitude of alkylbenzenes, aromatic compounds and monoterpenes, often involving novel biochemical reactions. In contrast, Az. indigens lineage members are diazotrophic endophytes lacking these catabolic capacities. It is proposed that species of the Az. evansii lineage should be classified in a novel genus, Aromatoleum gen. nov. Finally, based on the literature and new growth, DNA-DNA hybridization and proteomic data, the following five new species are proposed: Aromatoleum aromaticum sp. nov. (type strain EbN1 T =DSM 19018 T =LMG 30748 T and strain pCyN1=DSM 19016=LMG 31004), Aromatoleum petrolei sp. nov. (type strain ToN1 T =DSM 19019 T =LMG 30746 T ), Aromatoleumbremense sp. nov. (type strain PbN1 T =DSM 19017 T =LMG 31005 T ), Aromatoleum toluolicum sp. nov. (type strain T T =DSM 19020 T =LMG 30751 T ) and Aromatoleum diolicum sp. nov. (type strain 22Lin T =DSM 15408 T =LMG 30750 T ).

Candida auris was first reported in an ear swab from Japan in 2009; it then promptly spread over five continents and turned into a global nosocomial problem. The main challenges faced by many researchers are the mis -identification by conventional methods in clinical laboratories and failure in treatment. About 90% of C. auris strains are intrinsically resistant to fluconazole (FLU), and it is developing resistance to multiple classes of available antifungals. Echinocandins are the most potent class of antifungals against C. auris ; however, reduced susceptibility to one or many echinocandin drugs has been recently observed. Thus, the main issues addressed in this paper are the fast and accurate identification of C. auris derived from Sabouraud dextrose agar and blood culture bottles as well as the rapid antifungal susceptibility test by MALDI-TOF MS. This study successfully identified all isolates of C. auris ( n = 50) by MALDI-TOF MS, with an average log score of ≥ 2. An accuracy of 100% was found on both agar plate and blood culture bottles. MALDI Biotyper antibiotic susceptibility test-rapid assay (MBT ASTRA) was used for rapid antifungal susceptibility testing (AFST). A comparison between MBT ASTRA and the Clinical and Laboratory Standards Institute guidelines (CLSI) detected a sensitivity and specificity of 100% and 98% for anidulafungin, and 100% and 95.5% for micafungin, respectively. A categorical agreement of 98% and 96% was calculated for the two methods. For caspofungin, sensitivity and specificity of 100 and 73% were found, respectively, with a categorical agreement of 82%. MBT ASTRA has the great potential to detect C. auris isolates non-susceptible against echinocandin antifungals within 6 h, which makes it a promising candidate for AFST in clinical laboratories in the future.

Mass spectrometry imaging (MSI) of neuropeptides has become a well-established method with the ability to combine spatially resolved information from immunohistochemistry with peptidomics information from mass spectrometric analysis. Several studies have conducted MSI of insect neural tissues; however, these studies did not detect neuropeptide complements in manners comparable to those of conventional peptidomics. The aim of our study was to improve sample preparation so that MSI could provide comprehensive and reproducible neuropeptidomics information. Using the cockroach retrocerebral complex, the presented protocol produces enhanced coverage of neuropeptides at 15 μm spatial resolution, which was confirmed by parallel analysis of tissue extracts using electrospray-ionization MS. Altogether, more than 100 peptide signals from 15 neuropeptide-precursor genes could be traced with high spatial resolution. In addition, MSI spectra confirmed differential prohormone processing and distinct neuropeptide-based compartmentalization of the retrocerebral complex. We believe that our workflow facilitates incorporation of MSI in neuroscience-related topics, including the study of complex neuropeptide interactions within the CNS.

Phycotoxins and mycotoxins, such as paralytic shellfish poisoning toxins, type A trichothecenes, and aflatoxins are among the most toxic low molecular weight toxins associated with human poisoning incidents through the consumption of naturally contaminated food. Therefore, there is an utmost need for rapid and sensitive on-site detection systems. Herein, an electrochemical biochip for fast detection of saxitoxin, T-2 toxin as well as aflatoxin M1 and their corresponding congeners, respectively, using a portable and fully automated detection platform (pBDi, portable BioDetector integrated) was developed. Toxin analysis is facilitated upon the biochip via an indirect competitive immunoassay using toxin-specific antibodies combined with anti-idiotypic antibodies. The developed biochips enable detection in the low ng/mL-range within 17 min. Moreover, the assays cover a wide linear working range of 2-3 orders of magnitude above the limit of detection with an inter-chip coefficient of variation lower than 15%. The broad specificity of the employed antibodies which react with a large number of congeners within the respective toxin group allows efficient screening of contaminated samples for the presence of these low molecular weight toxins. With respect to the analysis of human urine samples, we focused here on the detection of saxitoxin, HT-2 toxin, and aflatoxin M1, all known as biomarkers of acute toxin exposure. Overall, it was proved that the developed biochip assays can be used to rapidly and reliably identify severe intoxications caused by these low molecular weight toxins.

Introduction: Antibiotic resistant bacteria are a growing concern worldwide. Extended-spectrum β-lactamases (ESBL) represent the most common resistance mechanism of Gram-negative bacteria against β-lactams, underlining the need for adequate diagnostic methods that provide reliable information in the shortest time possible. AmpC, a less prevalent but increasingly relevant class of β-lactamases, pose an additional challenge as their detection is complex. Here, we present an ESBL and AmpC screening panel employing the MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA). Materials and Methods: Four reference strains recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were used to develop the panel, which was further validated on 50 clinical Enterobacterales isolates resistant to third generation cephalosporins. The panel relies on the synergistic effect between ESBL and/or AmpC β-lactamase inhibitors and cephalosporins, which indicates β-lactamase production. Microdroplets containing the tested microorganism, cephalosporins in different concentrations and inhibitors were pipetted onto an MBT Biotarget and incubated for 3 or 4 h at 35 ± 1°C. Afterward, the liquid medium was removed and the material adhered to the spots was analyzed by MALDI-TOF MS. Synergy was detected by determining and comparing the minimum inhibitory concentrations of the tested cephalosporins with and without β-lactamase inhibitors. Data were interpreted following a diagnostic algorithm proposed by EUCAST in order to establish a final diagnosis. In comparison, PCR, broth microdilution (BMD) and combination disk tests (CDT) were performed. Results: Compared to the PCR results, the following positive and negative percent agreement values (PPA/NPA) were obtained for each resistance mechanism: ESBL, 94.44/100%; AmpC, 94.44/93.75% and ESBL+AmpC, 100/100%. These results, obtained after 4 h of incubation, were comparable with those of BMD and showed a higher accuracy than CDT. Discussion: We propose a novel phenotypic method for detection of ESBL and AmpC β-lactamases in Enterobacterales that provides reliable results in a short time, representing a promising alternative to the diagnostic techniques currently available. This easy-to-perform approach has potential for being implemented in routine laboratories, contributing to the further diversification of mass spectrometry technology into other fields such as antibiotic resistance testing.

Purpose: Identification of proteolytic peptides from matrix-assisted laser desorption/ionization (MALDI) imaging remains a challenge. The low fragmentation yields obtained using in situ post source decay impairs identification. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an alternative to in situ MS/MS, but leads to multiple identification candidates for a given mass. The authors propose to use LC-MS/MS-based biomarker discovery results to reliably identify proteolytic peptides from MALDI imaging., Experimental Design: The authors defined m/z values of interest for high grade squamous intraepithelial lesion (HSIL) by MALDI imaging. In parallel the authors used data from a biomarker discovery study to correlate m/z from MALDI imaging with masses of peptides identified by LC-MS/MS in HSIL. The authors neglected candidates that were not significantly more abundant in HSIL according to the biomarker discovery investigation., Results: The authors assigned identifications to three m/z of interest. The number of possible identifiers for MALDI imaging m/z peaks using LC-MS/MS-based biomarker discovery studies was reduced by about tenfold compared using a single LC-MS/MS experiment. One peptide identification candidate was validated by immunohistochemistry., Conclusion and Clinical Relevance: This concept combines LC-MS/MS-based quantitative proteomics with MALDI imaging and allows reliable peptide identification. Public datasets from LC-MS/MS biomarker discovery experiments will be useful to identify MALDI imaging m/z peaks., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: To present matrix-assisted laser desorption/ionization (MALDI) imaging as a powerful method to highlight various tissue compartments., Experimental Design: Formalin-fixed paraffin-embedded (FFPE) tissue of a uterine cervix, a pancreas, a duodenum, a teratoma, and a breast cancer tissue microarray (TMA) are analyzed by MALDI imaging and by immunohistochemistry (IHC). Peptide images are visualized and analyzed using FlexImaging and SCiLS Lab software. Different histological compartments are compared by hierarchical cluster analysis., Results: MALDI imaging highlights tissue compartments comparable to IHC. In cervical tissue, normal epithelium can be discerned from intraepithelial neoplasia. In pancreatic and duodenal tissues, m/z signals from lymph follicles, vessels, duodenal mucosa, normal pancreas, and smooth muscle structures can be visualized. In teratoma, specific m/z signals to discriminate squamous epithelium, sebaceous glands, and soft tissue are detected. Additionally, tumor tissue can be discerned from the surrounding stroma in small tissue cores of TMAs. Proteomic data acquisition of complex tissue compartments in FFPE tissue requires less than 1 h with recent mass spectrometers., Conclusion and Clinical Relevance: The simultaneous characterization of morphological and proteomic features in the same tissue section adds proteomic information for histopathological diagnostics, which relies at present on conventional hematoxylin and eosin staining, histochemical, IHC and molecular methods., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: To facilitate the transition of MALDI-MS Imaging (MALDI-MSI) from basic science to clinical application, it is necessary to analyze formalin-fixed paraffin-embedded (FFPE) tissues. The aim is to improve in situ tryptic digestion for MALDI-MSI of FFPE samples and determine if similar results would be reproducible if obtained from different sites., Experimental Design: FFPE tissues (mouse intestine, human ovarian teratoma, tissue microarray of tumor entities sampled from three different sites) are prepared for MALDI-MSI. Samples are coated with trypsin using an automated sprayer then incubated using deliquescence to maintain a stable humid environment. After digestion, samples are sprayed with CHCA using the same spraying device and analyzed with a rapifleX MALDI Tissuetyper at 50 µm spatial resolution. Data are analyzed using flexImaging, SCiLS, and R., Results: Trypsin application and digestion are identified as sources of variation and loss of spatial resolution in the MALDI-MSI of FFPE samples. Using the described workflow, it is possible to discriminate discrete histological features in different tissues and enabled different sites to generate images of similar quality when assessed by spatial segmentation and PCA., Conclusions and Clinical Relevance: Spatial resolution and site-to-site reproducibility can be maintained by adhering to a standardized MALDI-MSI workflow., (© 2018 The Authors. Proteomics - Clinical Application published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: To develop a mass spectrometry imaging (MSI) based workflow for extracting m/z values related to putative protein biomarkers and using these for reliable tumor classification., Experimental Design: Given a list of putative breast and ovarian cancer biomarker proteins, a set of related m/z values are extracted from heterogeneous MSI datasets derived from formalin-fixed paraffin-embedded tissue material. Based on these features, a linear discriminant analysis classification model is trained to discriminate the two tumor types., Results: It is shown that the discriminative power of classification models based on the extracted features is increased compared to the automatic training approach, especially when classifiers are applied to spectral data acquired under different conditions (instrument, preparation, laboratory)., Conclusions and Clinical Relevance: Robust classification models not confounded by technical variation between MSI measurements are obtained. This supports the assumption that the classification of the respective tumor types is based on biological rather than technical differences, and that the selected features are related to the proteomic profiles of the tumor types under consideration., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: To define proteomic differences between pancreatic ductal adenocarcinoma (pDAC) and pancreatic neuroendocrine tumor (pNET) by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI)., Experimental Design: Ninety-three pDAC and 126 pNET individual tissues are assembled in tissue microarrays and analyzed by MALDI MSI. The cohort is separated in a training (52 pDAC and 83 pNET) and validation set (41 pDAC and 43 pNET). Subsequently, a linear discriminant analysis (LDA) model based on 46 peptide ions is performed on the training set and evaluated on the validation cohort. Additionally, two liver metastases and a whole slide of pDAC are analyzed by the same LDA algorithm., Results: Classification of pDAC and pNET by the LDA model is correct in 95% (39/41) and 100% (43/43) of patients in the validation cohort, respectively. The two liver metastases and the whole slide of pDAC are also correctly classified in agreement with the histopathological diagnosis., Conclusion and Clinical Relevance: In the present study, a large dataset of pDAC and pNET by MALDI MSI is investigated, a class prediction model that allowed separation of both entities with high accuracy is developed, and differential peptide peaks with potential diagnostic, prognostic, and predictive values are highlighted., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Accurate and timely mycobacterial species identification is imperative for successful diagnosis, treatment, and management of disease caused by nontuberculous mycobacteria (NTM). The current most widely utilized method for NTM species identification is Sanger sequencing of one or more genomic loci, followed by BLAST sequence analysis. MALDI-TOF MS offers a less expensive and increasingly accurate alternative to sequencing, but the commercially available assays used in clinical mycobacteriology cannot differentiate between Mycobacterium intracellulare and Mycobacterium chimaera , two closely related potentially pathogenic species of NTM that are members of the Mycobacterium avium complex (MAC). Because this differentiation of MAC species is challenging in a diagnostic setting, Bruker has developed an improved spectral interpretation algorithm to differentiate M. chimaera and M. intracellulare based on differential spectral peak signatures. Here, we utilize a set of 185 MAC isolates that have been characterized using rpoB locus sequencing followed by whole genome sequencing in some cases, to test the accuracy of the Bruker subtyper software to identify M. chimaera ( n = 49) and M. intracellulare ( n = 55). 100% of the M. intracellulare and 82% of the M. chimaera isolates were accurately identified using the MALDI Biotyper algorithm. This subtyper module is available with the MALDI Biotyper Compass software and offers a promising mechanism for rapid and inexpensive species determination for M. chimaera and M. intracellulare .

In bottom-up proteomics, peptides are separated by liquid chromatography with elution peak widths in the range of seconds, whereas mass spectra are acquired in about 100 microseconds with time-of-flight (TOF) instruments. This allows adding ion mobility as a third dimension of separation. Among several formats, trapped ion mobility spectrometry (TIMS) is attractive because of its small size, low voltage requirements and high efficiency of ion utilization. We have recently demonstrated a scan mode termed parallel accumulation - serial fragmentation (PASEF), which multiplies the sequencing speed without any loss in sensitivity (Meier et al. , PMID: 26538118). Here we introduce the timsTOF Pro instrument, which optimally implements online PASEF. It features an orthogonal ion path into the ion mobility device, limiting the amount of debris entering the instrument and making it very robust in daily operation. We investigate different precursor selection schemes for shotgun proteomics to optimally allocate in excess of 100 fragmentation events per second. More than 600,000 fragmentation spectra in standard 120 min LC runs are achievable, which can be used for near exhaustive precursor selection in complex mixtures or accumulating the signal of weak precursors. In 120 min single runs of HeLa digest, MaxQuant identified more than 6,000 proteins without matching to a library and with high quantitative reproducibility (R > 0.97). Online PASEF achieves a remarkable sensitivity with more than 2,500 proteins identified in 30 min runs of only 10 ng HeLa digest. We also show that highly reproducible collisional cross sections can be acquired on a large scale (R > 0.99). PASEF on the timsTOF Pro is a valuable addition to the technological toolbox in proteomics, with a number of unique operating modes that are only beginning to be explored., (© 2018 Meier et al.)

Bacteroides fragilis is a frequent anaerobic pathogen and can cause severe infections. Resistance to carbapenems, associated with the cfiA gene encoded carbapenemase, represents an emerging problem. To date, no rapid methods are available to detect and confirm this resistance mechanism in routine laboratories, and the missed recognition of carbapenemase-producing strains can lead to therapeutic failures. In this study we have investigated a whole MALDI-TOF MS-based workflow to detect carbapenemase-producing B. fragilis, using the largest set of B. fragilis clinical isolates ever tested. The presence of the cfiA gene was predicted by MALDI subtyping into Division I (cfiA-negative) or Division II (cfiA-positive). The carbapenemase activity in cfiA-positive strains was confirmed by a MALDI-TOF MS imipenem hydrolysis assay (MBT STAR-Carba, Bruker Daltonik, Germany), that was further used for a characterization of the strains in terms of cfiA expression level. The validity of MALDI subtyping was verified by PCR for the cfiA gene, while results of MALDI hydrolysis assay were compared to conventional methods for susceptibility testing and carbapenemase detection (Carba-NP and disk diffusion synergy test). A genetic analysis of the IS elements upstream cfiA was performed, for the evaluations regarding the expression level of cfiA. A total of 5300 B. fragilis isolates (406 from Bologna, Italy, and 4894 from Dortmund, Germany) were identified and subtyped by MALDI-TOF MS, yielding 41/406 (10.1%) strains from Bologna and 374/4894 (7.6%) from Dortmund to belong to Division II. Molecular verification by PCR for the cfiA gene on a subset of strains confirmed the MALDI typing results in all cases (sensitivity and specificity of 100%). MBT STAR-Carba assay detected the carbapenemase activity in all of the 70 cfiA-carrying strains tested. Moreover, it allowed distinct separation into slow (59) and fast (11) imipenem hydrolyzers corresponding to cfiA expression levels as well as to low or high MICs for carbapenems, respectively. Among the 11 cfiA-positive strains with high carbapenem MIC, only 7 harboured IS elements upstream the carbapenemase gene showing low expression level as well. The MALDI-TOF MS-based workflow was superior to the currently available phenotypic methods for carbapenemase detection as it proved to be more sensitive and accurate than Carba NP and disk diffusion synergy test. The whole MALDI-TOF MS-based workflow allows an accurate identification of B. fragilis clinical strains with reliable classification into Division I/II, and confirmation of the carbapenemase-production, together with estimation of carbapenemase activity, within less than 2 h. This may be of particular interest for early therapeutical decisions in life-threatening infections., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

KPC-producing Klebsiella pneumoniae represents a severe public health concern worldwide. The rapid detection of these isolates is of fundamental importance for the adoption of proper antibiotic treatment and infection control measures, and new applications of MALDI-TOF MS technology fit this purpose. In this study, we present a full MALDI-based approach to detect plasmid-encoded KPC-producing strains, accomplished by the automated detection of a KPC-specific peak (at 11,109 m/z) by a specific algorithm integrated into the MALDI Biotyper system (Bruker Daltonik), and the confirmation of carbapenemase activity by STAR-Carba imipenem hydrolysis assay. A total of 6209 K. pneumoniae isolates from Italy and Germany were investigated for the presence of the KPC-related peak, and a subset of them ( n = 243) underwent confirmation of carbapenemase activity by STAR-Carba assay. The novel approach was further applied directly to positive blood culture bottles ( n = 204), using the bacterial pellet obtained with Sepsityper kit (Bruker Daltonik). The novel approach enabled a reliable and very fast detection of KPC-producing K. pneumoniae strains, from colonies as well as directly from positive blood cultures. The automated peak detection enabled the instant detection of KPC-producing K. pneumoniae during the routine identification process, with excellent specificity (100%) and a good sensitivity (85.1%). The sensitivity is likely mainly related to the prevalence of the specific plasmid harboring clones among all the KPC-producing circulating strains. STAR-Carba carbapenemase confirmation showed 100% sensitivity and specificity, both from colonies and from positive blood cultures.

Human antibodies of the IgG2 subclass exhibit complex inter-chain disulfide bonding patterns that result in three structures, namely A, A/B, and B. In therapeutic applications, the distribution of disulfide isoforms is a critical product quality attribute because each configuration affects higher order structure, stability, isoelectric point, and antigen binding. The current standard for quantification of IgG2 disulfide isoform distribution is based on chromatographic or electrophoretic techniques that require additional characterization using mass spectrometry (MS)-based methods to confirm disulfide linkages. Detailed characterization of the IgG2 disulfide linkages often involve MS/MS approaches that include electrospray ionization or electron-transfer dissociation, and method optimization is often cumbersome due to the large size and heterogeneity of the disulfide-bonded peptides. As reported here, we developed a rapid LC-MALDI-TOF/TOF workflow that can both identify the IgG2 disulfide linkages and provide a semi-quantitative assessment of the distribution of the disulfide isoforms. We established signature disulfide-bonded IgG2 hinge peptides that correspond to the A, A/B, and B disulfide isoforms and can be applied to the fast classification of IgG2 isoforms in heterogeneous mixtures.

Mixtures of pollen grains of three different species (Corylus avellana, Alnus cordata, and Pinus sylvestris) were investigated by matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-TOF imaging MS). The amount of pollen grains was reduced stepwise from > 10 to single pollen grains. For sample pretreatment, we modified a previously applied approach, where any additional extraction steps were omitted. Our results show that characteristic pollen MALDI mass spectra can be obtained from a single pollen grain, which is the prerequisite for a reliable pollen classification in practical applications. MALDI imaging of laterally resolved pollen grains provides additional information by reducing the complexity of the MS spectra of mixtures, where frequently peak discrimination is observed. Combined with multivariate statistical analyses, such as principal component analysis (PCA), our approach offers the chance for a fast and reliable identification of individual pollen grains by mass spectrometry. Graphical Abstract ᅟ.

Klebsiella pneumoniae and related species are frequent causes of nosocomial infections and outbreaks. Therefore, quick and reliable strain typing is crucial for the detection of transmission routes in the hospital. The aim of this study was to evaluate Fourier transform infrared spectroscopy (FTIR) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as rapid methods for typing clinical Klebsiella isolates in comparison to whole-genome sequencing (WGS), which was considered the gold standard for typing and identification. Here, 68 clinical Klebsiella strains were analyzed by WGS, FTIR, and MALDI-TOF MS. FTIR showed high discriminatory power in comparison to the WGS reference, whereas MALDI-TOF MS exhibited a low ability to type the isolates. MALDI-TOF mass spectra were further analyzed for peaks that showed high specificity for different Klebsiella species. Phylogenetic analysis revealed that the Klebsiella isolates comprised three different species: K. pneumoniae , K. variicola , and K. quasipneumoniae Genome analysis showed that MALDI-TOF MS can be used to distinguish K. pneumoniae from K. variicola due to shifts of certain mass peaks. The peaks were tentatively identified as three ribosomal proteins (S15p, L28p, L31p) and one stress response protein (YjbJ), which exhibit amino acid differences between the two species. Overall, FTIR has high discriminatory power to recognize the clonal relationship of isolates, thus representing a valuable tool for rapid outbreak analysis and for the detection of transmission events due to fast turnaround times and low costs per sample. Furthermore, specific amino acid substitutions allow the discrimination of K. pneumoniae and K. variicola by MALDI-TOF MS., (Copyright © 2018 American Society for Microbiology.)

The recently developed direct-on-target microdroplet growth assay (DOT-MGA) allows rapid universal antimicrobial susceptibility testing (AST) using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Here, we investigated a direct application of this method on positive blood cultures (BCs) for the acceleration of sepsis diagnostics. Blood samples spiked with meropenem-nonsusceptible and meropenem-susceptible Enterobacterales isolates were inoculated into Bactec Plus Aerobic/F bottles and incubated in the Bactec automated system. Positive-BC broth was processed using four different methods, filtration/dilution, dilution, lysis/centrifugation, and differential centrifugation. For both dilution-based methods, AST was performed from 1:100, 1:1,000, and 1:10,000 dilutions of positive-BC broth in cation-adjusted Mueller-Hinton broth (CA-MHB). For both centrifugation-based methods, a 0.5 McFarland standard turbidity suspension was prepared from a bacterial pellet and adjusted to a final inoculum of 5 × 10 5 CFU/ml in CA-MHB. Six-microliter microdroplets with or without meropenem at the breakpoint concentration were spotted in triplicate onto a MALDI-TOF MS target, followed by incubation in a humidity chamber for 3 or 4 h and subsequent broth removal. Spectra were evaluated by MALDI Biotyper software. The test was considered valid if the growth control without antibiotic achieved an identification score of ≥1.7. For samples with meropenem, successful identification (score, ≥1.7) was interpreted as a nonsusceptible result, whereas failed identification (score, <1.7) defined susceptibility. The best test performance was achieved with the lysis/centrifugation method after a 4-h incubation. At this time point, 96.3% validity, 91.7% sensitivity, and 100% specificity were reached. This study demonstrated the feasibility and accuracy of a rapid DOT-MGA from positive BCs. Parallel to susceptibility determination, this method provides simultaneous species identification., (Copyright © 2018 American Society for Microbiology.)

In order to understand the distribution of the main secondary metabolites found in Olea europaea L., eight different samples (olive leaf, stem, seed, fruit skin and pulp, as well as virgin olive oil, olive oil obtained from stoned and dehydrated fruits and olive seed oil) coming from a Picudo cv. olive tree were analyzed. All the experimental conditions were selected so as to assure the maximum coverage of the metabolome of the samples under study within a single run. The use of LC and GC with high resolution MS (through different ionization sources, ESI and APCI) and the annotation strategies within MetaboScape 3.0 software allowed the identification of around 150 compounds in the profiles, showing great complementarity between the evaluated methodologies. The identified metabolites belonged to different chemical classes: triterpenic acids and dialcohols, tocopherols, sterols, free fatty acids, and several sub-types of phenolic compounds. The suitability of each platform and polarity (negative and positive) to determine each family of metabolites was evaluated in-depth, finding, for instance, that LC-ESI-MS (+) was the most efficient choice to ionize phenolic acids, secoiridoids, flavonoids and lignans and LC-APCI-MS was very appropriate for pentacyclic triterpenic acids (MS (-)) and sterols and tocopherols (MS (+)). Afterwards, a semi-quantitative comparison of the selected matrices was carried out, establishing their typical features (e.g., fruit skin was pointed out as the matrix with the highest relative amounts of phenolic acids, triterpenic compounds and hydroxylated fatty acids, and seed oil was distinctive for its high relative levels of acetoxypinoresinol and tocopherols).

Pasteurella multocida is notorious for its role as an opportunistic pathogen in infectious bronchopneumonia, the economically most important disease facing cattle industry and leading indication for antimicrobial therapy. To rationalize antimicrobial use, avoiding imprudent use of highly and critically important antimicrobials for human medicine, availability of a rapid antimicrobial susceptibility test is crucial. The objective of the present study was to design a MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA) procedure for tetracycline resistance detection in P. multocida. This procedure was validated on 100 clinical isolates with MIC-gradient strip test, and a comparison with disk diffusion was made. Sensitivity and specificity of the MBT-ASTRA procedure were 95.7% (95% confidence interval (CI) = 89.8-101.5) and 100% (95% CI = 100-100), respectively, classifying 98% of the isolates correctly after only three hours of incubation. Sensitivity and specificity of disk diffusion were 93.5% (95% CI = 86.3-100.6) and 96.3% (95% CI = 91.3-101.3) respectively, classifying 95% of the isolates correctly. In conclusion, this MBT-ASTRA procedure has all the potential to fulfil the need for a rapid and highly accurate tetracycline susceptibility testing in P. multocida to rationalize antimicrobial use in outbreaks of bronchopneumonia in cattle or other clinical presentations across species.

1. TBA-354 was a promising antitubercular compound with activity against both replicating and static Mycobacterium tuberculosis (M.tb), making it the focal point of many clinical trials conducted by the TB Alliance. However, findings from these trials have shown that TBA-354 results in mild signs of reversible neurotoxicity; this left the TB Alliance with no other choice but to stop the research. 2. In this study, mass spectrometric methods were used to evaluate the pharmacokinetics and spatial distribution of TBA-354 in the brain using a validated liquid chromatography tandem-mass spectrometry (LCMS/MS) and mass spectrometric imaging (MSI), respectively. Healthy female Sprague-Dawley rats received intraperitoneal (i.p.) doses of TBA-354 (20 mg/kg bw). 3. The concentrationtime profiles showed a gradual absorption and tissue penetration of TBA-354 reaching the C max at 6 h post dose, followed by a rapid elimination. MSI analysis showed a time-dependent drug distribution, with highest drug concentration mainly in the neocortical regions of the brain. 4. The distribution of TBA-354 provides a possible explanation for the motor dysfunction observed in clinical trials. These results prove the importance of MSI as a potential tool in preclinical evaluations of suspected neurotoxic compounds.

Coral reef ecosystems are highly sensitive to microbial activities that result from dissolved organic matter (DOM) enrichment of their surrounding seawater. However, the response to particulate organic matter (POM) enrichment is less studied. In a microcosm experiment, we tested the response of bacterioplankton to a pulse of POM from the mass-spawning of Orbicella franksi coral off the Caribbean coast of Panama. Particulate organic carbon (POC), a proxy measurement for POM, increased by 40-fold in seawater samples collected during spawning; 68% degraded within 66 h. The elevation of multiple hydrolases presumably solubilized the spawn-derived POM into DOM. A carbon budget constructed for the 275 µM of degraded POC showed negligible change to the concentration of dissolved organic carbon (DOC), indicating that the DOM was readily utilized. Fourier transform ion cyclotron resonance mass spectrometry shows that the DOM pool became enriched with heteroatom-containing molecules, a trend that suggests microbial alteration of organic matter. Our sensitivity analysis demonstrates that bacterial carbon demand could have accounted for a large proportion of the POC degradation. Further, using bromodeoxyuridine immunocapture in combination with 454 pyrosequencing of the 16S ribosomal RNA gene, we surmise that actively growing bacterial groups were the primary degraders. We conclude that coral gametes are highly labile to bacteria and that such large capacity for bacterial degradation and alteration of organic matter has implications for coral reef health and coastal marine biogeochemistry.

The Bruker MALDI Biotyper® method utilizes matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for the rapid and accurate identification and confirmation of Gram-negative bacteria from select media types. The alternative method was evaluated using nonselective and selective agars to identify Cronobacter spp., Salmonella spp., and select Gram-negative bacteria. Results obtained by the Bruker MALDI Biotyper were compared to the traditional biochemical methods as prescribed in the appropriate reference methods. Two collaborative studies were organized, one in the United States focusing on Cronobacter spp. and other Gram-negative bacteria, and one in Europe focusing on Salmonella spp. and other Gram-negative bacteria. Fourteen collaborators from seven laboratories located within the United States participated in the first collaborative study for Cronobacter spp. Fifteen collaborators from 15 service laboratories located within Europe participated in the second collaborative study for Salmonella spp. For each target organism (either Salmonella spp. or Cronobacter spp.), a total of 24 blind-coded isolates were evaluated. In each set of 24 organisms, there were 16 inclusivity organisms (Cronobacter spp. or Salmonella spp.) and 8 exclusivity organisms (closely related non-Cronobacter spp. and non-Salmonella spp. Gram-negative organisms). After testing was completed, the total percentage of correct identifications from each agar type for each strain was determined at a percentage of 100.0% to the genus level for the Cronobacter study and a percentage of 100.0% to the genus level for the Salmonella study. For both non-Cronobacter and non-Salmonella organisms, a percentage of 100.0% was correctly identified. The results indicated that the alternative method produced equivalent results when compared to the confirmatory procedures specified by each reference method.

The Bruker MALDI Biotyper® method utilizes matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for the rapid and accurate confirmation and identification of Gram-positive bacteria from select media types. This alternative method was evaluated using nonselective and selective agar plates to identify and confirm Listeria monocytogenes, Listeria species, and select Gram-positive bacteria. Results obtained by the Bruker MALDI Biotyper were compared with the traditional biochemical methods as prescribed in the appropriate reference method standards. Sixteen collaborators from 16 different laboratories located within the European Union participated in the collaborative study. A total of 36 blind-coded isolates were evaluated by each collaborator. In each set of 36 organisms, there were 16 L. monocytogenes strains, 12 non-monocytogenes Listeria species strains, and 8 additional Gram-positive exclusivity strains. After testing was completed, the total percentage of correct identifications (to both genus and species level) and confirmation from each agar type for each strain was determined at a percentage of 99.9% to the genus level and 98.8% to the species level. The results indicated that the alternative method produced equivalent results when compared with the confirmatory procedures specified by each reference method.

Mass spectrometry imaging (MSI) has provided many results with translational character, which still have to be proven robust in large patient cohorts and across different centers. Although formalin-fixed paraffin-embedded (FFPE) specimens are most common in clinical practice, no MSI multicenter study has been reported for FFPE samples. Here, we report the results of the first round robin MSI study on FFPE tissues with the goal to investigate the consequences of inter- and intracenter technical variation on masking biological effects. A total of four centers were involved with similar MSI instrumentation and sample preparation equipment. A FFPE multi-organ tissue microarray containing eight different types of tissue was analyzed on a peptide and metabolite level, which enabled investigating different molecular and biological differences. Statistical analyses revealed that peptide intercenter variation was significantly lower and metabolite intercenter variation was significantly higher than the respective intracenter variations. When looking at relative univariate effects of mass signals with statistical discriminatory power, the metabolite data was more reproducible across centers compared to the peptide data. With respect to absolute effects (cross-center common intensity scale), multivariate classifiers were able to reach on average > 90% accuracy for peptides and > 80% for metabolites if trained with sufficient amount of cross-center data. Overall, our study showed that MSI data from FFPE samples could be reproduced to a high degree across centers. While metabolite data exhibited more reproducibility with respect to relative effects, peptide data-based classifiers were more directly transferable between centers and therefore more robust than expected. Graphical abstract ᅟ.

The incidence of candidemia caused by Candida albicans and Candida glabrata is constantly increasing and is accompanied by the rising use of the few available antifungals. The widespread use of echinocandins and azoles for the treatment of invasive candidemia has enhanced the development of antifungal resistance, resulting in an increasing health care problem. Hence, the rapid detection of resistant strains is required. This study aimed to evaluate the detection of C. albicans and C. glabrata strains resistant to caspofungin by the matrix-assisted laser desorption ionization Biotyper antibiotic susceptibility test rapid assay (MBT ASTRA). This novel semiquantitative technique facilitates the detection of caspofungin-resistant strains within 6 h. MBT ASTRA results were compared to the data obtained by the use of Clinical and Laboratory Standards Institute (CLSI) guidelines. Clinical isolates of C. albicans ( n = 58) and C. glabrata ( n = 57) were analyzed by MBT ASTRA and the CLSI microdilution method. Antifungal susceptibility testing against caspofungin by the CLSI microdilution method classified the C. albicans isolates into 36 susceptible and 22 resistant strains and the C. glabrata isolates into 5 susceptible, 33 resistant, and 19 intermediate strains. For C. albicans , the comparison of MBT ASTRA and the CLSI method revealed an excellent categorical agreement of 100%. A sensitivity and a specificity between MBT ASTRA and the CLSI microdilution method of 94% and 80%, respectively, were detected for C. glabrata strains, based on categorical agreement. In conclusion, the results obtained by MBT ASTRA indicate that this is a very promising approach for the rapid detection of Candida isolates resistant to caspofungin., (Copyright © 2018 American Society for Microbiology.)

Pre-clinical and clinical studies are now beginning to demonstrate the high potential of cell therapies in enhancing muscle regeneration. We previously demonstrated functional benefit after the transplantation of autologous bone marrow mesenchymal stromal cells (MSC-TX) into a severe muscle crush trauma model. Despite our increasing understanding of the molecular and cellular mechanisms underlying MSC's regenerative function, little is known about the local molecular alterations and their spatial distribution within the tissue after MSC-TX. Here, we used MALDI imaging mass spectrometry (MALDI-IMS) in combination with multivariate statistical strategies to uncover previously unknown peptide alterations within severely injured skeletal muscles. Our analysis revealed that very early molecular alterations in response to MSC-TX occur largely in the region adjacent to the trauma and only to a small extent in the actual trauma region. Using "bottom up" mass spectrometry, we subsequently identified the proteins corresponding to the differentially expressed peptide intensity distributions in the specific muscle regions and used immunohistochemistry to validate our results. These findings extend our current understanding about the early molecular processes of muscle healing and highlights the critical role of trauma adjacent tissue during the early therapeutic response upon treatment with MSC.

Multiple sulfatase deficiency (MSD) is a fatal, inherited lysosomal storage disorder characterized by reduced activities of all sulfatases in patients. Sulfatases require a unique post-translational modification of an active-site cysteine to formylglycine that is catalyzed by the formylglycine-generating enzyme (FGE). FGE mutations that affect intracellular protein stability determine residual enzyme activity and disease severity in MSD patients. Here, we show that protein disulfide isomerase (PDI) plays a pivotal role in the recognition and quality control of MSD-causing FGE variants. Overexpression of PDI reduces the residual activity of unstable FGE variants, whereas inhibition of PDI function rescues the residual activity of sulfatases in MSD fibroblasts. Mass spectrometric analysis of a PDI+FGE variant covalent complex allowed determination of the molecular signature for FGE recognition by PDI. Our findings highlight the role of PDI as a disease modifier in MSD, which may also be relevant for other ER-associated protein folding pathologies., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objectives: We aimed to develop a universal phenotypic method, which allows easy and rapid antimicrobial susceptibility testing independently of underlying resistance mechanisms., Methods: We established a novel direct-on-target microdroplet growth assay for the detection of antibiotic resistance within a few hours, which is based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The microorganisms were incubated with and without meropenem in nutrient broth as microdroplets directly on MALDI-TOF MS target. Subsequently, broth was separated from microbial cells by contacting the microdroplets with an absorptive material. The microorganisms grown in the presence of antibiotic were detected by MALDI-TOF MS. A total of 24 Klebsiella pneumoniae and 24 Pseudomonas aeruginosa isolates were used to assess performance for detection of meropenem resistance. The microdroplet volumes investigated were 2, 4, 6, 8 and 10 μL., Results: The best performance was achieved using 6-μL microdroplets. Applying this volume, all growth controls were successfully detected (definition of valid test), and all isolates were correctly categorized as susceptible or non-susceptible after an 18-h incubation. For K. pneumoniae, rate of valid tests, sensitivity and specificity all reached 100% after a 4-h incubation of 6-μL microdroplets. Using the same microdroplet volume for P. aeruginosa, incubation for 5 h resulted in 83.3% of valid tests with 100% sensitivity and 100% specificity., Conclusions: We demonstrated easy, rapid and accurate resistance detection using carbapenem-resistant Gram-negative bacteria as an example. Our technology is suitable for automatization and expandable to further applications, e.g. simultaneous testing of multiple antibiotics as well as resistance determination directly from clinical samples., (Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Top-Down approaches have an extremely high biological relevance, especially when it comes to biomarker discovery, but the necessary pre-fractionation constraints are not easily compatible with the robustness requirements and the size of clinical sample cohorts. We have demonstrated that intact protein profiling studies could be run on UHR-Q-ToF with limited pre-fractionation (Schmit et al., 2017) [1]. The dataset presented herein is an extension of this research. Proteoforms known to play a role in the pathophysiology process of Alzheimer's disease were identified as candidate biomarkers. In this article, mass spectrometry performance of these candidates are demonstrated.

Thanks to proteomics investigations, our vision of the role of different protein isoforms in the pathophysiology of diseases has largely evolved. The idea that protein biomarkers like tau, amyloid peptides, ApoE, cystatin, or neurogranin are represented in body fluids as single species is obviously over-simplified, as most proteins are present in different isoforms and subjected to numerous processing and post-translational modifications. Measuring the intact mass of proteins by MS has the advantage to provide information on the presence and relative amount of the different proteoforms. Such Top-Down approaches typically require a high degree of sample pre-fractionation to allow the MS system to deliver optimal performance in terms of dynamic range, mass accuracy and resolution. In clinical studies, however, the requirements for pre-analytical robustness and sample size large enough for statistical power restrict the routine use of a high degree of sample pre-fractionation. In this study, we have investigated the capacities of current-generation Ultra-High Resolution Q-Tof systems to deal with high complexity intact protein samples and have evaluated the approach on a cohort of patients suffering from neurodegenerative disease. Statistical analysis has shown that several proteoforms can be used to distinguish Alzheimer disease patients from patients suffering from other neurodegenerative disease., Significance: Top-down approaches have an extremely high biological relevance, especially when it comes to biomarker discovery, but the necessary pre-fractionation constraints are not easily compatible with the robustness requirements and the size of clinical sample cohorts. We have demonstrated that intact protein profiling studies could be run on UHR-Q-ToF with limited pre-fractionation. The proteoforms that have been identified as candidate biomarkers in the-proof-of concept study are derived from proteins known to play a role in the pathophysiology process of Alzheimer disease., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In the present study 12 Arcanobacterium phocae strains isolated from fur animals in Finland, including foxes, minks and Finnraccoons, could successfully be identified phenotypically, by matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) and genotypically by sequencing 16S rDNA and phocaelysin (PHL) encoding gene phl. The PHL of all 12 A. phocae strains in the present study and reference strains A. phocae DSM 10002 T and A. phocae DSM 10003 displayed, as typical members of the cholesterol dependent cytolysin-group of toxins, the variant undecapeptide sequence EATGLAWDPWW which appeared to be most closely related to arcanolysin of Arcanobacterium haemolyticum and pyolysin of Trueperella pyogenes. In addition, gene phl could be determined with a newly designed loop-mediated isothermal amplification (LAMP) assay. The detection of mass spectra by MALDI-TOF MS and the LAMP assay based on gene phl might help to reliably identify A. phocae in future and also elucidate the role this species plays in infections of fur animals., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Introduction: The introduction of the MALDI Biotyper in laboratories substantially changed microbiology practice, this has been called a revolution. The system accelerated diagnostic while costs were reduced and accuracy was increased. In just a few years MALDI-TOF MS became the first-line identification tool for microorganisms. Ten years after its introduction, more than 2000 MALDI Biotyper systems are installed in laboratories which are performing routine diagnostic, and the number is still increasing. Areas covered: This article summarises changes in clinical microbiology introduced by the MALDI Biotyper and its effects, as it has been published in peer reviewed articles found in PubMed. Further, the potential of novel developments to increase the value of the system is described. Expert commentary: The MALDI Biotyper has significantly improved clinical microbiology in the area of microorganism identification. Now new developments and applications, e.g. for typing and resistance testing, might further increase its value in clinical microbiology. The systems might get the central diagnostic analyser which is getting integrated into the widely automated microbiology laboratories of the future.

Since food spoilage by yeasts causes high economic losses, fast and accurate identifications of yeasts associated with food and food-related products are important for the food industry. In this study the efficiency of the matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify food related yeasts was evaluated. A CBS in-house MALDI-TOF MS database was created and later challenged with a blinded test set of 146 yeast strains obtained from food and food related products. Ninety eight percent of the strains were correctly identified with log score values>1.7. One strain, Mrakia frigida, gained a correct identification with a score value<1.7. Two strains could not be identified at first as they represented a mix of two different species. These mixes were Rhodotorula babjevae with Meyerozyma caribbica and Clavispora lusitaniae with Debaryomyces hansenii. After separation, all four species could be correctly identified with scores>1.7. Ambiguous identifications were observed due to two incorrect reference mass spectra's found in the commercial database BDAL v.4.0, namely Candida sake DSM 70763 which was re-identified as Candida oleophila, and Candida inconspicua DSM 70631 which was re-identified as Pichia membranifaciens. MALDI-TOF MS can distinguish between most of the species, but for some species complexes, such as the Kazachstania telluris and Mrakia frigida complexes, MALDI-TOF MS showed limited resolution and identification of sibling species was sometimes problematic. Despite this, we showed that the MALDI-TOF MS is applicable for routine identification and validation of foodborne yeasts, but a further update of the commercial reference databases is needed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Formerly only referred to as a subspecies (T. mentagrophytes var. quinckeanum), T. quinckeanum once again constitutes a distinct species according to the updated taxonomy of dermatophytes., Patients and Methods: During routine diagnostic tests conducted at the Mycology Laboratory, Mölbis, Germany, between 11/2013 to 1/2017 (three years and three months), all specimens sent in were examined for T. quinckeanum. Molecular biology methods employed included: 1) DNA hybridization (PCR ELISA), 2) gene sequencing of the ITS region and TEF-1α, and 3) in some cases, MALDI-TOF mass spectrometry., Results: Overall, 62 strains of T. quinckeanum were found. Sixty-eight percent of patients were female; 43 % were children and adolescents (≤ 19 years of age). Cats were a frequent source of infection. Sequencing of all 62 strains revealed a concordance of 100 % with T. quinckeanum sequences contained in the NCBI database. Isolates analyzed by MALDI-TOF mass spectrometry showed specific spectra., Conclusions: In Germany, the zoophilic dermatophyte T. quinckeanum currently appears to be more prevalent than expected. T. quinckeanum strains were isolated from children and adults with dermatomycosis and tinea capitis. Sources of infection with T. quinckeanum include small rodents (mice), horses, and - remarkably commonly -  cats. Given that unequivocal morphological identification of this dermatophyte is not always possible, molecular methods have to be employed in the diagnosis., (© 2017 Deutsche Dermatologische Gesellschaft (DDG). Published by John Wiley & Sons Ltd.)

This study validates the analysis of polycyclic aromatic hydrocarbons (PAHs) in microgram levels of particulate matter (PM) collected on filters by two low-flow rate, real-time monitors, microPEM™ and microAeth ® . Particle-associated PAHs were analyzed by a coupling of a gas chromatograph to a sensitive, atmospheric-pressure laser ionization-mass spectrometer. Air particulate samples were collected over the course of one or two days in the living room of a fourth-floor apartment in New York City. Three types of samplers, the two aforementioned personal samplers and a high-flow rate pump (4 liters per minute), were operated side by side, and three samples of each type were collected during each sampling period. Intrasampler agreement as measured by relative standard deviation (RSD) was within 1% to 18%. After background subtraction, total PAH measured by all three sampler types had good agreement (R=0.99). This ability to accurately characterize personal PAH exposure in archived filters collected by these real-time samplers could provide additional important PAH exposure information that can benefit many environmental health studies using these monitors.

The present study was designed to characterize phenotypically and genotypically a Trueperella pyogenes strain isolated from a brain abscess of an adult roebuck (Capreolus capreolus). The species identity could be confirmed by phenotypical investigations, by MALDI-TOF MS analysis, and by sequencing the 16S ribosomal RNA (rRNA) gene, the 16S-23S rRNA intergenic spacer region (ISR); by sequencing the target genes rpoB, gap, and tuf; and by detection of T. pyogenes chaperonin-encoding gene cpn60 with a previously developed loop-mediated isothermal amplification (LAMP) assay. The T. pyogenes strain could additionally be characterized by PCR-mediated amplification of several known and putative virulence factor-encoding genes which revealed the presence of the genes plo encoding pyolysin and nanH and nanP encoding neuraminidases; the genes fimA, fimC, and fimE encoding the fimbrial subunits FimA, FimC, and FimE; and the gene cbpA encoding collagen-binding protein CbpA. The present data give a detailed characterization of a T. pyogenes strain isolated from a brain abscess of a roebuck. However, the route of infection of the roebuck remains unclear.