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Seven genotypically distinct strains assigned to the genus Erysipelothrix were isolated in different laboratories from several animal sources. Strain D17_0559-3-2-1T and three further strains were isolated from samples of duck, pig and goose. The strains had >99 % 16S rRNA gene sequence similarity to each other and to strain VA92-K48T and two further strains isolated from samples of medical leech and a turtle. The closest related type strains to the seven strains were those of Erysipelothrix inopinata (96.74 %) and Erysipelothrix rhusiopathiae (95.93 %). Average nucleotide identity, amino acid identity and in silico DNA–DNA hybridization results showed that the strains represented two separate novel species. One further phylogenetically distinct strain (165301687T) was isolated from fox urine. The strain had highest 16S rRNA gene sequence similarity to the type strains of Erysipelothrix tonsillarum (95.67 %), followed by Erysipelothrix piscisicarius (95.58 %) and Erysipelothrix larvae (94.22 %) and represented a further novel species. Chemotaxonomic and physiological data of the novel strains were assessed, but failed to unequivocally differentiate the novel species from existing members of the genus. MALDI-TOF MS data proved the discrimination of at least strain 165301687T from all currently described species. Based on the presented phylogenomic and physiological data, we propose three novel species, Erysipelothrix anatis sp. nov. with strain D17_0559-3-2-1T (=DSM 111258T= CIP 111884T=CCM 9044T) as type strain, Erysipelothrix aquatica sp. nov. with strain VA92-K48T (=DSM 106012T=LMG 30351T=CIP 111492T) as type strain and Erysipelothrix urinaevulpis sp. nov. with strain 165301687T (=DSM 106013T= LMG 30352T= CIP 111494T) as type strain.

The increasing development of antibiotic resistance in bacteria has been a major problem for years, both in human and veterinary medicine. Prophylactic measures, such as the use of vaccines, are of great importance in reducing the use of antibiotics in livestock. These vaccines are mainly produced based on formaldehyde inactivation. However, the latter damages the recognition elements of the bacterial proteins and thus could reduce the immune response in the animal. An alternative inactivation method developed in this work is based on gentle photodynamic inactivation using carbon nanodots (CNDs) at excitation wavelengths λ

In Germany, infections due to the zoophilic dermatophyte Trichophyton (T.) species of Arthroderma benhamiae are being more frequently diagnosed. The source of infection of this emerging pathogen overlaps with that of the zoophilic species T. interdigitale. The most common source are guinea pigs. T. species of Arthroderma benhamiae causes inflammatory dermatophytosis in children and adolescents. In addition to tinea capitis, it may cause both tinea corporis, tinea manus and frequently tinea faciei. In Germany, T. species of Arthroderma benhamiae is a frequent zoophilic dermatophyte, which in regions is probably more frequent than Microsporum canis. The mycological identification of the isolates with their yellow stained colonies is based on their macroscopic and microscopic features. However, some exhibit colony features consistent with those of T. interdigitale. These strains only can be identified unambiguously by means of molecular techniques. Using detection methods such as PCR-ELISA or real-time PCR, the dermatophyte can be identified directly from clinical material. Sequencing of the internal transcribed spacer region (ITS) of the ribosomal DNA has been approved as culture confirmation test for T. species of Arthroderma benhamiae. In addition, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) is useful. Widespread dermatophytosis due to T. species of Arthroderma benhamiae, in particular of tinea capitis, requires oral antifungal agents. Terbinafine is most effective, alternatives are fluconazole and itraconazole.

Zusammenfassung In Deutschland kommen seit einigen Jahren Infektionen durch den zoophilen Dermatophyten Trichophyton (T.) Spezies von Arthroderma benhamiae vor. Das Reservoir fur diesen neuen Erreger – ein emerging pathogen – uberlappt mit dem des zoophilen T. interdigitale. Insbesondere Meerschweinchen sind Carrier. T. Spezies von Arthroderma benhamiae verursacht eine entzundliche Tinea bei Kindern und Jugendlichen. Neben der Tinea capitis werden Tinea corporis und Tinea manus verursacht, vor allem jedoch die Tinea faciei. T. Spezies von Arthroderma benhamiae ist in Deutschland ein haufiger zoophiler Dermatophyt, in manchen Regionen haufiger als Microsporum canis. Die Identifizierung der Isolate mit gelb gefarbten Kolonien ist anhand makro- und mikroskopischer Merkmale moglich. Ein Teil der Isolate weist jedoch Koloniemerkmale auf, welche mit denen von T. interdigitale ubereinstimmen. Diese Stamme lassen sich nur mittels molekularer Methoden identifizieren. Mit einem PCR-ELISA oder real-time PCR kann der Dermatophyt direkt im klinischen Material nachgewiesen werden. Als Kulturbestatigungstest hat sich die Sequenzierung der internal transcribed spacer Region (ITS) der ribosomalen DNA bewahrt. Auch die matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) ist dafur geeignet. Die Behandlung von ausgedehnten Dermatophytosen durch T. Spezies von Arthroderma benhamiae, insbesondere der Tinea capitis, erfolgt mit oralen Antimykotika, am besten Terbinafin; Alternativen sind Fluconazol und Itraconazol.

Altogether 285 dermatophyte isolates of 21 different species - including both Trichophyton rubrum and T. interdigitale, but also eight additional Trichophyton species, Microsporum canis and seven other Microsporum species, as well as Epidermophyton floccosum and Arthroderma spp. - were analyzed using Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and the AnagnosTec 'SARAMIS' (Spectral Archiving and Microbial Identification System) software. In addition, sequence analysis of the internal transcribed spacer (ITS) of the ribosomal DNA was performed for a high number of the tested strains. Sufficient agreement was found between the results obtained with standard identification methods and those with the MALDI-TOF MS for species identification of dermatophytes. A mass spectra database was constructed which contained the species identifications of all 285 isolates. The results were confirmed for 164 of the isolates by sequence analysis of the internal transcribed spacer (ITS) of the ribosomal DNA. Statistical analysis of all 285 dermatophyte strains showed that conventional identification matched the results of MALDI-TOF MS for 78.2% of the isolates tested. In the case of the 164 isolates for which the identifications were confirmed by PCR, the results of their conventional diagnosis and MALDI-TOF MS were in agreement for only 68.9 % (113 of 164 strains) of the test isolates. In contrast, there was agreement of 99.3 % or 98.8 % in the identifications obtained with PCR and MALDI-TOF MS techniques (283/285 or 162/164). The two exceptions were isolates that proved to be T. violaceum which could not be identified by the MALDI-TOF MS technique. In conclusion, the MALDI-TOF mass spectroscopy represents a fast and very specific method for species differentiation of dermatophytes grown in culture.

Clinical microbiology is a conservative laboratory exercise where base technologies introduced in the 19th century remained essentially unaltered. High-tech mass spectrometry (MS) has changed that. Within a few years following its adaptation to microbiological diagnostics, MS has been introduced, embraced, and broadly accepted by clinical microbiology laboratories throughout the world as an innovative tool for definitive bacterial species identification. Herein, we review the current state of the art with respect to this exciting new technology and discuss potential future applications.

Species identification of yeasts is based on biochemical (e.g. API ID 32 C®, bioMerieux) and molecular biological approaches. As an alternative to DNA-dependent methods, mass spectral analysis based identification of micro-organisms has become increasingly recognized. In a number of studies, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been applied for the rapid classification and identification of micro-organisms. In this study, the applicability of MALDI-TOF MS for identifying yeasts isolated from dermatological patients was analysed and compared with the results from the API ID 32 C® system. Furthermore, sequencing the internal transcribed spacer (ITS) regions of the ribosomal DNA was employed as reference method. Candida (C.) albicans was isolated in 41.9% of all cases, C. parapsilosis in 20.3%, C. glabrata in 10.8%, and C. krusei in 6, 8.1%. Rarely isolated yeasts were Candida colliculosa, famata, guilliermondii, lusitaniae, and tropicalis as well as Geotrichum candidum, Rhodotorula mucilaginosa and Trichosporon mucoides. The MALDI TOF results were equal to the results gained by ITS sequence analysis in 94%, whereas API ID 32 C® provided the correct diagnosis in 84.3% (of all cases). This lower identification rate is mostly referable to frequent misidentifications of C. krusei as C. inconspicua/norvegensis,Candida tropicalis, or Geotrichum capitatum. In contrast, all C. krusei strains were correctly identified by MALDI TOF MS. In conclusion, species identification by MALDI-TOF MS was proven to be consistent with ITS sequence analysis; the technique has a resolving power comparatively as high as ITS sequence analysis.

The Streptococcus bovis/equinus complex is a heterogeneous group within the group D streptococci with important clinical relevance regarding infective endocarditis, sepsis and colon carcinoma. The taxonomic identification of species and sub-species of this complex, by the standard methods remains difficult. In the present study, we compared the cluster analysis of 88 strains of species of the S. bovis/equinus complex by sequence analysis of the manganese-dependent superoxide dismutase gene (sodA) and by Matrix Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS). We observed a high congruence of strain grouping by MALDI-TOF MS in comparison with sodA sequence analyses, demonstrating the accuracy and reliability of MALDI-TOF MS in comparison to DNA sequence-based method. By generating mass spectra for each species and sub-species, we were able to discriminate all members of the S. bovis/equinus complex. Furthermore, we demonstrated reliable identifications to the species level by MALDI-TOF MS, independently of cultivation conditions.

Gram-positive anaerobic cocci (GPAC) are part of the commensal microbiota of humans and are a phylogenetically heterogeneous group of organisms. To evaluate the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of GPAC, a database was constructed, using reference strains of commonly encountered GPAC and clinical isolates of which the sequence of the 16S rRNA gene was determined. Subsequently, the database was validated by identifying 107 clinical isolates of GPAC. Results were compared with the identifications obtained by 165 sequencing or fluorescent in situ hybridization (FISH). Strains belonging to the same species grouped together, in most cases, by MALDI-TOF MS analyses. Strains with sequence similarities less than 98% to their closest relatives, formed clusters distinct from recognized species in the MALDI-TOF MS dendrogram and, therefore could not be identified. These strains probably represent new species. Only three clinical isolates (2 strains of Finegoldia magna and 1 strain of Anaerococcus vaginalis) could not be identified. For all the other GPAC strains (96/107), reliable identifications were obtained. Therefore, we concluded that MALDI-TOF MS is an excellent tool for the identification of phylogenetically heterogeneous groups of micro-organisms such as GPAC. (C) 2010 Elsevier GmbH. All rights reserved.

Microcystins (MCs) are cyclic heptapeptides, which are the most abundant toxins produced by cyanobacteria in freshwater. The phytoplankton of many freshwater lakes in Eastern Africa is dominated by cyanobacteria. Less is known, however, on the occurrence of MC producers and the production of MCs. Twelve Ugandan freshwater habitats ranging from mesotrophic to hypertrophic conditions were sampled in May and June of 2004 and April of 2008 and were analyzed for their physicochemical parameters, phytoplankton composition, and MC concentrations. Among the group of the potential MC-producing cyanobacteria, Anabaena (0-10(7) cells ml(-1)) and Microcystis (10(3)-10(7) cells ml(-1)) occurred most frequently and dominated in eutrophic systems. A significant linear relationship (n = 31, r(2) = 0.38, P0.001) between the Microcystis cell numbers and MC concentration (1.3-93 fg of MC cell(-1)) was observed. Besides [MeAsp(3), Mdha(7)]-MC-RR, two new MCs, [Asp(3)]-MC-RY and [MeAsp(3)]-MC-RY, were isolated and their constitution was assigned by LC-MS(2). To identify the MC-producing organism in the water samples, (i) the conserved aminotransferase domain part of the mcyE gene that is indicative of MC production was amplified by general primers and cloned and sequenced, and (ii) genus-specific primers were used to amplify the mcyE gene of the genera Microcystis, Anabaena, and Planktothrix. Only mcyE genotypes that are indicative of Microcystis sp. were obtained via the environmental cloning approach (337 bp, 96.1-96.7% similarity to the Microcystis aeruginosa strain PCC7806). Accordingly, only the mcyE primers, which are specific for Microcystis, revealed PCR products. We concluded that Microcystis is the major MC-producer in Ugandan freshwater.

Although mycoses are among the most common diseases worldwide, infections with Fusarium spp. occur only rarely. Mostly patients suffering from underlying immune deficiency are infected with this mould, resulting in a considerably decreasing prognosis. In immunocompromised patients, cutaneous manifestations are more often associated with Fusarium sp. than with Candida sp. or Aspergillus sp. We describe one patient with acute lymphoblastic leukaemia, who was first treated with chemotherapy after GMALL protocol 07/03. After relapse, the patient was successfully transplanted in second remission with a human leukocyte antigen (HLA)-matched unrelated peripheral blood stem cell graft. Ten months later, the patient died from respiratory insufficiency and recurrence of leukaemia. Previously, Aspergillus antigen was detected in blood. In the latter course, disseminated papules appeared. One of these was examined histologically and mycologically. Conventional cultural diagnostics led to the diagnosis of a fusariosis, further supported by internal transcribed spacer (ITS) sequencing and matrix assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF) mass spectrometry, both determining the isolated strain as Fusarium proliferatum, which is a very infrequent pathogen within this genus. Our investigations underline the potential of MALDI-TOF MS based identification of Fusarium species as an innovative, time and cost efficient alternative to ITS sequencing.

Microcystis is a well-known cyanobacterial genus frequently producing hepatotoxins named microcystins. Toxin production is encoded by microcystin genes (mcy). This study aims (i) to relate the mcy occurrence in individual colonies to the presence of microcystin, (ii) to assess whether morphological characteristics (morphospecies) are related to the occurrence of mcy genes, and (iii) to test whether there are geographical variations in morphospecies specificity and abundance of mcy genes. Individual colonies of nine different European countries were analysed by (1) morphological characteristics, (2) PCR to amplify a gene region within mcyA and mcyB indicative for microcystin biosynthesis, (3) matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) to detect microcystins. Almost one hundred percent of the colonies predicted to produce microcystins by PCR analysis were found to contain microcystins. A high similarity in microcystin variants in the different colonies selected from lakes across Europe was demonstrated. The different morphospecies varied in the frequency with which they contained mcy genes. Most colonies (>75%) of M. aeruginosa and M. botrys contained the mcy genes, whereas < or = 20% of the colonies identified as M. ichthyoblabe and M. viridis gave a PCR product of the mcy genes. No colonies of M. wesenbergii gave a PCR product of either mcy gene. In addition, a positive relationship was found between the size of the colony and the frequency of those containing the mcy genes. It is concluded that the analysis of morphospecies is indicative for microcystin production, although the quantitative analysis of microcystin concentrations in water remains indispensable for hazard control.

Two depsipeptide metabolites, scyptolin A and B, were reported recently from the axenically grown terrestrial cyanobacterium Scytonema hofmanni PCC 7110. A related, novel depsipeptide was isolated from this Scytonema and designated hofmannolin. The amino acid analysis in context with infrared, mass and 1 H/ 13 C-NMR spectroscopies revealed a cyclic depsipeptide structure of M r 1073 belonging to the class of cyanopeptolins. Two peculiar features distinguish hofmannolin from other cyanopeptolins: O -methylated tyrosine forms the sixth moiety from the amino terminus, and the N-terminus is blocked by 2-hydroxy-3-methyl-valeric acid, a residue that has not yet been reported as a component in other cyanopeptolins. Preliminary assays of peptidase inhibitory and antimicrobial activities suggested negligible bioactivities for hofmannolin.

The occurrence of toxic blooms of cyanobacteria in due course of the eutrophication of most inland waters has become an increasingly important issue of scientific and public health concern. One objective of many field studies is to assess the predictability of bloom toxicity. Here we present data of a four-year monitoring of microcystins in lake Muggelsee together with data on spatial distribution and diversity of the predominant toxin-producer Microcystis sp. Microcystins were detectable in all seasons when Microcystis -biovolumes were detectable with seasonal maximum concentrations of 1-3 μg/L. In spatially resolved samples, the microcystin concentrations spanned more than an order of magnitude between up-wind and down-wind sites with highest concentrations at the latter ones. The bloom development of Microcystis and Aphanizomen was significantly correlated to the ratio of DIN: SRP and it is discussed whether this reflects a causal relationship or rather similar trends in both variables related to third one, like water temperature. The microcystin content of Microcystis was negatively correlated to Microcystis abundance, thus blooms were relatively less toxic during their peak. A metabolic typing of strains isolated from a single bloom sample revealed that the Microcystis bloom was composed of a multitude of toxin-producing and non-producing strains that differed in their production of microcystins and other non-ribosomal peptides. The number of distinguishable chemolypes clearly exceeded the number of microscopically determined morphotypes. Given the high clonal diversity of the Microcystis community we argue that a quantitative prediction of bloom toxicity is not possible without a better understanding of the molecular ecology of Microcystis and that an avoidance of toxic blooms of Microcystis is best to be done by appropriate restoration measures.

This paper describes the purification and characterization of microviridin J, a newly discovered metabolite of Microcystis that causes a lethal molting disruption in Daphnia spp., upon ingestion of living cyanobacterial cells. Microviridin J consists of an acetylated chain of 13 amino acids arranged in three rings and two side chains. Unlike other known isoforms of microviridin, microviridin J contains arginine that imparts a unique solution conformation characterized by proximal hydrophobic interactions between Arg and other regions of the molecule. This eventually results in the formation and stabilization of an additional ring system. Microviridin J potently inhibits porcine trypsin, bovine chymotrypsin, and daphnid trypsin-like proteases. The activity against trypsin is most likely due to Arg and its distinctive conformational interactions. Overall, the data presented for microviridin J emphasize once again the ability of cyanobacteria to produce numerous and potent environmental toxins.

Recent advances in analytical chemistry have provided an array of techniques to analyze cyanotoxins in a variety of samples. Mass spectral analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a reliable and rapid tool to detect and identify microcystin variants in very small samples such as single Microcystis colonies. Mass signals presumptive of microcystin variants can be characterized further and identified by postsource-decay fragmentation and comparison of observed fragment spectra with theoretical ones or with those of pure reference compounds. By analyzing Microcystis colonies from water blooms, the high metabolic diversity among individual colonies in the communities could be established. For monitoring microcystins in environmental samples, MALDI-TOF MS can provide considerable support to HPLC by identifying microcystin variants not available as purified standards, especially in Planktothrix-dominated blooms in which demethylated variants are predominant. A short description of the principles of MALDI-TOF MS is provided, followed by examples of its application and a discussion of its potential use in cyanotoxin research.

In Germany, infections due to the zoophilic dermatophyte Trichophyton (T.) species of Arthroderma benhamiae are being more frequently diagnosed. The source of infection of this emerging pathogen overlaps with that of the zoophilic species T. interdigitale. The most common source are guinea pigs. T. species of Arthroderma benhamiae causes inflammatory dermatophytosis in children and adolescents. In addition to tinea capitis, it may cause both tinea corporis, tinea manus and frequently tinea faciei. In Germany, T. species of Arthroderma benhamiae is a frequent zoophilic dermatophyte, which in regions is probably more frequent than Microsporum canis. The mycological identification of the isolates with their yellow stained colonies is based on their macroscopic and microscopic features. However, some exhibit colony features consistent with those of T. interdigitale. These strains only can be identified unambiguously by means of molecular techniques. Using detection methods such as PCR-ELISA or real-time PCR, the dermatophyte can be identified directly from clinical material. Sequencing of the internal transcribed spacer region (ITS) of the ribosomal DNA has been approved as culture confirmation test for T. species of Arthroderma benhamiae. In addition, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) is useful. Widespread dermatophytosis due to T. species of Arthroderma benhamiae, in particular of tinea capitis, requires oral antifungal agents. Terbinafine is most effective, alternatives are fluconazole and itraconazole.

Extended spectrum of β-lactam (ESBL) resistance of Klebsiella pneumoniae has become an increasing problem in hospital infections. Typing of isolates is important to establish the intrahospital surveillance of resistant clones. In this study, the discriminatory potential of randomly amplified polymorphic DNA and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analyses were compared with multilocus sequence typing (MLST) by using 17 β-lactam-resistant K. pneumoniae isolates of different genotypes. MLST alleles were distributed in 8 sequence types (STs). Among ESBL strains of the same ST, the presence of different β-lactamase genes was common. RAPD band patterns also revealed 8 types that corresponded to MLST-defined genotypes in 15 out of 17 cases. MALDI-TOF analysis could differentiate 5 clusters of strains. The results of this work show that RAPD may be usable as a rapid screening method for the intrahospital surveillance of K. pneumoniae, allowing a discrimination of clonally related strains. MALDI-TOF-based typing was not strongly corresponding to genotyping and warrants further investigation.

© 2014 Gomila et al. The Achromobacter is a genus in the family Alcaligenaceae, comprising fifteen species isolated from different sources, including clinical samples. The ability to detect and correctly identify Achromobacter species, particularly A. xylosoxidans, and differentiate them from other phenotypically similar and genotypically related Gram-negative, aerobic, non-fermenting species is important for patients with cystic fibrosis (CF), as well as for nosocomial and other opportunistic infections. Traditional phenotypic profile-based analyses have been demonstrated to be inadequate for reliable identifications of isolates of Achromobacter species and genotypic-based assays, relying upon comparative 16S rRNA gene sequence analyses are not able to insure definitive identifications of Achromobacter species, due to the inherently conserved nature of the gene. The uses of alternative methodologies to enable high-resolution differentiation between the species in the genus are needed. A comparative multi-locus sequence analysis (MLSA) of four selected 'house-keeping' genes (atpD, gyrB, recA, and rpoB) assessed the individual gene sequences for their potential in developing a reliable, rapid and cost-effective diagnostic protocol for Achromobacter species identifications. The analysis of the type strains of the species of the genus and 46 strains of Achromobacter species showed congruence between the cluster analyses derived from the individual genes. The MLSA gene sequences exhibited different levels of resolution in delineating the validly published Achromobacter species and elucidated strains that represent new genotypes and probable new species of the genus. Our results also suggested that the recently described A. spritinus is a later heterotypic synonym of A. marplatensis. Strains were analyzed, using whole-cell Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF MS), as an alternative phenotypic profile-based method with the potential to support the identifications determined by the genotypic DNA sequencebased MLSA. The MALDI-TOF MS data showed good accordance in strain groupings and identifications by the MLSA data. Copyright, Margarita Gomila is the recipient of a postdoctoral contract from the University of the Balearic Islands funded by the Spanish Ministry of Education, Culture and Sports through the International Excellence Campus Program. Margarita Gomila was supported for exchange to the CCUG through fellowships from the Spanish Ministry of Education and Science, by means of the José Castillejo Program (2008) and from a FEMS Research Fellowship 2009-1. This work was supported also by funding from Västra Götaland Region (Sweden) projects ALFGBG-210591, VGFOUREG-157801 and VGFOUREG-232981, and from Spanish MINECO, through projects CGL2011-24318 and Consolider CSD2009-00006

Microcystin is a potent inhibitor of eukaryotic protein phosphatases and has been implicated in causing hepatotoxicity to humans and animals worldwide. It is produced primarily by the bloom-forming cyanobacterium Microcystis aeruginosa, although the function of the peptide in this micro-organism is unknown. In this study, a microcystin-related protein, MrpA, was identified using a microcystin-lacking mutant of M. aeruginosa, PCC 7806. Comparative two-dimensional protein electrophoresis showed that MrpA was strongly expressed in wild-type PCC 7806, but was not detectable in the mcyB mutant. MrpA showed similarity to the RhiA protein from Rhizobium leguminosarum, which is encoded by the rhiABC operon and controlled by quorum-sensing mediators. Sequencing of mrpA flanking regions in M. aeruginosa PCC 7806 revealed the presence of a rhiB homologue, mrpB, directly downstream of mrpA. Northern blot analyses of mrpA expression in cells exposed to different light conditions revealed a rapid decline of transcription under high light conditions. Most striking was a strong increase in transcript levels from cultures irradiated with blue light. The mrpA transcription level was strongly reduced in two independent microcystin-lacking mutants under all light conditions investigated.

Besides the most prominent peptide toxin, microcystin, the cyanobacteria Microcystis spp. have been shown to produce a large variety of other bioactive oligopeptides. We investigated for the first time the oligopeptide diversity within a natural Microcystis population by analyzing single colonies directly with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). The results demonstrate a high diversity of known cyanobacterial peptides such as microcystins, anabaenopeptins, microginins, aeruginosins, and cyanopeptolins, but also many unknown substances in the Microcystis colonies. Oligopeptide patterns were mostly related to specific Microcystis taxa. Microcystis aeruginosa (Kütz.) Kütz. colonies contained mainly microcystins, occasionally accompanied by aeruginosins. In contrast, microcystins were not detected in Microcystis ichthyoblabe Kütz.; instead, colonies of this species contained anabaenopeptins and/or microginins or unknown peptides. Within a third group, Microcystis wesenbergii (Kom.) Kom. in Kondr., chiefly a cyanopeptolin and an unknown peptide were found. Similar patterns, however, were also found in colonies which could not be identified to species level. The significance of oligopeptides as a chemotaxonomic tool within the genus Microcystis is discussed. It could be demonstrated that the typing of single colonies by MALDI-TOF MS may be a valuable tool for ecological studies of the genus Microcystis as well as in early warning of toxic cyanobacterial blooms.

Fifteen microcystins (MC) were isolated from field samples dominated by Microcystis spp., Planktothrix agardhii or Planktothrix rubescens using reversed phase high-performance liquid chromatography (HPLC). Characterization of microcystins was performed with amino acid analysis followed by fast atom bombardment mass spectrometry (FABMS), and/or matrix assisted laser desorption ionization mass spectrometry (MALDI-TOF MS). In blooms of Microcystis spp. major microcystins were usually MC-RR, MC-YR and MC-LR, while in one strain [D-Asp 3 ] MC-LR and [D-Asp 3 ] MC-RR were also quantitatively abundant. The main microcystins found in field samples of Planktothrix agardhii were [D-Asp 3 ] MC-RR followed by [D-Asp 3 ] MC-LR and a [D-Asp 3 ] variant of MC-HtyR. Planktothrix agardhii strain HUB 076 produced [D-Asp 3 ] MC-RR as a single component. In all Planktothrix rubescens populations [D-Asp 3 , Dhb 7 ] MC-RR was the most abundant microcystin. Furthermore we found that microcystin composition in field populations of single species scarcely changed over time as long as dominance of this species persisted.

Several bloom-forming cyanobacterial genera produce potent inhibitors of eukaryotic protein phosphatases called microcystins. Microcystins are hepatotoxic cyclic heptapeptides and are presumed to be synthesized non-ribosomally by peptide synthetases. We identified putative peptide synthetase genes in the microcystin-producing strain Microcystis aeruginosa PCC 7806. Non-hepatotoxic strains of M. aeruginosa lack these genes. Strain PCC 7806 was transformed to chloramphenicol resistance. The antibiotic resistance cassette insertionally inactivated a peptide synthetase gene of strain PCC 7806 as revealed by Southern hybridization and DNA amplification. This is the first report of genetic transformation and mutation, by homologous recombination, of a bloom-forming cyanobacterium. Chemical and enzymatic analyses, including high-performance liquid chromatography (HPLC), mass spectrometry, amino acid activation, and protein phosphatase inhibition, revealed the inability of derived mutant cells to produce any variant of microcystin while maintaining their ability to synthesize other small peptides. The disrupted gene therefore encodes a peptide synthetase (microcystin synthetase) that is specifically involved in the biosynthesis of microcystins. Our results confirm that microcystins are synthesized non-ribosomally and that a basic difference between toxic and non-toxic strains of M. aeruginosa is the presence of one or more genes coding for microcystin synthetases.

In the present study four Trueperella (T.) abortisuis strains isolated from an umbilical swab, two anal swabs and from a placenta after abortion of four pigs, respectively, could successfully be identified phenotypically, by MALDI-TOF MS analysis and genotypically by amplification and sequencing of 16S rRNA gene sequence and gene sodA encoding superoxide dismutase A as additional molecular target. All four T. abortisuis were isolated together with various other bacterial species indicating that the pathogenic importance of this novel species remains unclear. However, according to the literature and to the results of the present study T. abortisuis could be recovered from samples of animals in Japan and in different microbiological laboratories in Germany emphasizing its increasing importance.

Some members of the genus Candida are among the most common human fungal pathogens and cause serious diseases especially in immunocompromised people. A yeast was isolated from a blood culture from an immunocompromised cancer patient who suffered from acute pneumonia. The growth characteristics of the yeast on CHROMagar Candida were similar to those of Candida tropicalis , whereas the API ID 32C system identified the yeast as Candida silvicola . On the basis of the nucleotide divergence in the D1/D2 domain of the 26S nuclear rRNA (nrRNA) gene, as well as the internal transcribed spacer (ITS) domain of the nrRNA gene region, a new species, Candida pseudoaaseri sp. nov. with type strain VK065094 (CBS 11170 T ), which was found to be closely related to Candida aaseri , is proposed. While C. aaseri strains were susceptible to all tested antifungals, the new species is resistant to flucytosine and may also be distinguished from C. aaseri by its ability to assimilate l -rhamnose, whereas its colony morphology on CHROMagar Candida may be helpful for differentiation.

Species identification of yeasts is based on biochemical (e.g. API ID 32 C®, bioMérieux) and molecular biological approaches. As an alternative to DNA-dependent methods, mass spectral analysis based identification of micro-organisms has become increasingly recognized. In a number of studies, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been applied for the rapid classification and identification of micro-organisms. In this study, the applicability of MALDI-TOF MS for identifying yeasts isolated from dermatological patients was analysed and compared with the results from the API ID 32 C® system. Furthermore, sequencing the internal transcribed spacer (ITS) regions of the ribosomal DNA was employed as reference method. Candida (C.) albicans was isolated in 41.9% of all cases, C. parapsilosis in 20.3%, C. glabrata in 10.8%, and C. krusei in 6, 8.1%. Rarely isolated yeasts were Candida colliculosa, famata, guilliermondii, lusitaniae, and tropicalis as well as Geotrichum candidum, Rhodotorula mucilaginosa and Trichosporon mucoides. The MALDI TOF results were equal to the results gained by ITS sequence analysis in 94%, whereas API ID 32 C® provided the correct diagnosis in 84.3% (of all cases). This lower identification rate is mostly referable to frequent misidentifications of C. krusei as C. inconspicua/norvegensis,Candida tropicalis, or Geotrichum capitatum. In contrast, all C. krusei strains were correctly identified by MALDI TOF MS. In conclusion, species identification by MALDI-TOF MS was proven to be consistent with ITS sequence analysis; the technique has a resolving power comparatively as high as ITS sequence analysis.

Variations in the mass spectral profiles of multiple housekeeping proteins of 126 strains representing Salmonella enterica subsp. enterica (subspecies I), S. enterica subsp. salamae (subspecies II), S. enterica subsp. arizonae (subspecies IIIa), S. enterica subsp. diarizonae (subspecies IIIb), S. enterica subsp. houtenae (subspecies IV), and S. enterica subsp. indica (subspecies VI), and Salmonella bongori were analyzed to obtain a phylogenetic classification of salmonellae based on whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometric bacterial typing. Sinapinic acid produced highly informative spectra containing a large number of biomarkers and covering a wide molecular mass range (2,000 to 40,000 Da). Genus-, species-, and subspecies-identifying biomarker ions were assigned on the basis of available genome sequence data for Salmonella , and more than 200 biomarker peaks, which corresponded mainly to abundant and highly basic ribosomal or nucleic acid binding proteins, were selected. A detailed comparative analysis of the biomarker profiles of Salmonella strains revealed sequence variations corresponding to single or multiple amino acid changes in multiple housekeeping proteins. The resulting mass spectrometry-based bacterial classification was very comparable to the results of DNA sequence-based methods. A rapid protocol that allowed identification of Salmonella subspecies in minutes was established.

Identification of microorganisms, specifically of vegetative cells and spores, by intact cell mass spectrometry (ICMS) is an emerging new technology. The technique provides specific biomarker profiles which can be employed for bacterial identification at the genus, species, or even at the subspecies level holding the potential to serve as a rapid and sensitive identification technique in clinical or food microbiology and also for sensitive detection of biosafety level (BSL) 3 microorganisms. However, the development of ICMS as an identification technique for BSL-3 level microorganisms is hampered by the fact that no MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) compatible inactivation procedure for microorganisms, and particularly for bacterial endospores, has been evaluated so far. In this report we describe a new methodology for effective inactivation of microorganisms which is compatible with the analysis of microbial protein patterns by MALDI-TOF mass spectrometry. The main challenge of this work was to define the conditions that ensure microbial inactivation and permit at the same time comprehensive analysis of microbial protein patterns. Among several physical, chemical, and mechanical inactivation procedures, inactivation by trifluoroacetic acid (TFA) proved to be the best method in terms of bactericidal capacity and information content of the mass spectra. Treatment of vegetative cells by 80% TFA alone for 30 min assured complete inactivation of microbial cells under all conditions tested. For spore inactivation, the "TFA inactivation protocol" was developed which is a combination of TFA treatment with basic laboratory routines such as centrifugation and filtering. This MALDI-TOF/ICMS compatible sample preparation protocol is simple and rapid (30 min) and assures reliable inactivation of vegetative cells and spores of highly pathogenic (BSL-3) microorganisms.

Anatoxin-a (ANTX) and homoanatoxin-a (HANTX) are low molecular weight neurotoxic secondary amines of 165 and 179 Da, respectively. We applied matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) for the detection of ANTX and HANTX directly on lyophilized and fresh filaments of axenic strains of the genus Oscillatoria, using 2,5-dihydroxybenzoic acid as matrix and purified ANTX and HANTX as references. To counteract the span of low molecular mass ions (< m/z 1000) generated by the matrix, we induced the matrix-suppression effect to obtain high quality ANTX/HANTX MALDI signals. MALDI desorption/ionization of the matrix-ANTX and the matrix-HANTX generated protonated molecules [M+H](+) at m/z 166.12322 and 180.1372, respectively. The masses obtained from the analysis of lyophilized filaments of the ANTX-producer Oscillatoria sp. strain PCC 9240 (m/z 166.15) and of fresh filaments of the HANTX-producers Oscillatoria sp. strains PCC 6506 (m/z 180.1375), PCC 9029 (m/z 180.1334) and PCC 10111 (m/z 180.13996) corresponded to the protonated molecular ions of ANTX and HANTX, respectively. Therefore, the application of MALDI-TOF-MS for the detection of cyanobacterial anatoxins in clonal and axenic strains of the cyanobacterial culture collections worldwide may help to assess ANTX/HANTX incidence among cyanobacteria.

Identification of dermatophytes is currently performed based on morphological criteria and is increasingly supported by genomic sequence comparison. The present study evaluates an alternative based on the analysis of clinical fungal isolates by mass spectrometry. Samples originating from skin and nail were characterized morphologically and by sequencing the internal transcribed spacer 1 (ITS1), ITS2 and the 5.8S rDNA regions of the rDNA clusters. In a blind comparative study, samples were analyzed by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF MS). The mass spectra were compared to a database comprising of the spectral data of reference strains by applying the saramis software package. All fungal isolates belonging to the taxa Trichophyton rubrum, T. interdigitale, T. tonsurans, Arthroderma benhamiae and Microsporum canis were correctly identified, irrespective of host origin and pathology. To test the robustness of the approach, four isolates were grown on five different media and analyzed. Although the resulting mass spectra varied in detail, a sufficient number of signals were conserved resulting in data sets exploitable for unequivocal species identification. Taken together, the usually widespread dermatophytes can be identified rapidly and reliably by mass spectrometry. Starting from pure cultures, MALDI-TOF MS analysis uses very simple sample preparation procedures, and a single analysis is performed within minutes. Costs for consumables as well as preparation time are considerably lower than for PCR analysis.

The high metabolic diversity of potentially toxigenic cyanobacteria has been recently demonstrated for several bloom-forming genera. To understand the dynamics of cyanobacterial blooms and fluctuations in toxin content, techniques are required that allow the distinction of clones in field samples on a subspecific taxonomic level. We evaluated the possibility to analyze single filaments of a common toxigenic taxon, Planktothrix rubescens, by comparing peptide profiles obtained from HPLC/MS analyses of bulk biomass to mass spectra of single filaments for 11 clonal strains. For all strains a significant relationship of chromatogram peak area to mass spectrum peak height was found for individual peptides. Individual peptide's response in mass spectrometry compared to HPLC, however, differed, likely attributable to the presence of particular moieties with high proton affinities in the molecules. Thirty-four individual peptides detected in HPLC fractions of all strains could also be detected in the corresponding single filaments. In conclusion, MALDI-TOF MS analysis of single filaments is shown to be an efficient tool for the study of chemotype diversity and dynamics in field studies and laboratory experiments. The good correspondence between peak area and peak height in chromatograms and mass spectra, respectively, is promising with respect to the use of MALDI-TOF MS as a quantitative analytical tool for natural products studies and cyanotoxin research.

Nine Corallococcus isolates and three type strains of Corallococcus species were characterized by Intact Cell Mass Spectrometry using Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. The resulting phenetic clustering was compared to the phylogenetic grouping based upon sequences of two housekeeping genes. The three dendrograms of relatedness resembled each other in that the isolates were highly similar to the type strains of Corallococcus exiguus and Corallococcus coralloides, while Corallococcus macrosporus and Myxococcus xanthus were more distantly related. While certain pairs of organisms were recovered by spectrometry and genes sequence analysis, others were detected by two of the three approaches. The degree of similarity determined by sequence analysis of the two genes was not higher than that revealed by MALDI-TOF analysis. The results show that the spectral profile, consisting of about 25 to 45 masses ranging between 2 and 20 kDa, have indeed taxonomic significance, confirming literature data that ribosomal proteins and certain housekeeping proteins are responsible for the masses obtained. Provided the availability of a database of type strains, MALDI-TOF analysis of unknown strains appears to be a rapid and inexpensive method to taxonomically cluster environmental isolates, expanding the spectrum to strains other than those of medical importance predominantly investigated so far.

This paper describes the purification and characterization of microviridin J. a newly discovered metabolite of Microcystis that causes a lethal molting disruption in Daphnia spp., upon ingestion of living cyanobacterial cells. Microviridin J consists of an acetylated chain of 13 amino acids arranged in three rings and two side chains. Unlike other known isoforms of microviridin, microviridin J contains arginine that imparts a unique solution conformation characterized by proximal hydrophobic interactions between Arg and other regions of the molecule. This eventually results in the formation and stabilization of an additional ring system. Microviridin J potently inhibits porcine trypsin, bovine chymotrypsin, and daphnid trypsin-like proteases. The activity against trypsin is most likely due to Arg and its distinctive conformational interactions. Overall, the data presented for microviridin J emphasize once again the ability of cyanobacteria to produce numerous and potent environmental toxins.

Microcystins represent an extraordinarily large family of cyclic heptapeptide toxins that are nonribosomally synthesized by various cyanobacteria. Microcystins specifically inhibit the eukaryotic protein phosphatases 1 and 2A. Their outstanding variability makes them particularly useful for studies on the evolution of structure-function relationships in peptide synthetases and their genes. Analyses of microcystin synthetase genes provide valuable clues for the potential and limits of combinatorial biosynthesis. We have sequenced and analyzed 55.6 kb of the potential microcystin synthetase gene ( mcy ) cluster from the filamentous cyanobacterium Planktothrix agardhii CYA 126. The cluster contains genes for peptide synthetases ( mcyABC ), polyketide synthases (PKSs; mcyD ), chimeric enzymes composed of peptide synthetase and PKS modules ( mcyEG ), a putative thioesterase ( mcyT ), a putative ABC transporter ( mcyH ), and a putative peptide-modifying enzyme ( mcyJ ). The gene content and arrangement and the sequence of specific domains in the gene products differ from those of the mcy cluster in Microcystis , a unicellular cyanobacterium. The data suggest an evolution of mcy clusters from, rather than to, genes for nodularin (a related pentapeptide) biosynthesis. Our data do not support the idea of horizontal gene transfer of complete mcy gene clusters between the genera. We have established a protocol for stable genetic transformation of Planktothrix , a genus that is characterized by multicellular filaments exhibiting continuous motility. Targeted mutation of mcyJ revealed its function as a gene coding for a O -methyltransferase. The mutant cells produce a novel microcystin variant exhibiting reduced inhibitory activity toward protein phosphatases.

Background: Blooms of toxic cyanobacteria (blue-green algae) have become increasingly common in the surface waters of the world. Of the known toxins produced by cyanobacteria, the microcystins are the most significant threat to human and animal health. These cyclic peptides are potent inhibitors of eukaryotic protein phosphatases type 1 and 2A. Synthesized nonribosomally, the microcystins contain a number of unusual amino acid residues including the β-amino polyketide moiety Adda (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid). We have characterized the microcystin biosynthetic gene cluster from Microcystis aeruginosa PCC7806. Results: A cluster spanning 55 kb, composed of 10 bidirectionally transcribed open reading frames arranged in two putative operons (mcyA–C and mcyD–J) , has been correlated with microcystin formation by gene disruption and mutant analysis. Of the 48 sequential catalytic reactions involved in microcystin synthesis, 45 have been assigned to catalytic domains within six large multienzyme synthases/synthetases (McyA–E, G), which incorporate the precursors phenylacetate, malonyl-CoA, S -adenosyl-L-methionine, glutamate, serine, alanine, leucine, D-methyl-isoaspartate, and arginine. The additional four monofunctional proteins are putatively involved in O -methylation (McyJ), epimerization (McyF), dehydration (McyI), and localization (McyH). The unusual polyketide amino acid Adda is formed by transamination of a polyketide precursor as enzyme-bound intermediate, and not released during the process. Conclusions: This report is the first complete description of the biosynthesis pathway of a complex cyanobacterial metabolite. The enzymatic organization of the microcystin assembly represents an integrated polyketide–peptide biosynthetic pathway with a number of unusual structural and enzymatic features. These include the integrated synthesis of a β-amino-pentaketide precursor and the formation of β- and γ-carboxyl-peptide bonds, respectively. Other features of this complex system also observed in diverse related biosynthetic clusters are integrated C - and N -methyltransferases, an integrated aminotransferase, and an associated O -methyltransferase and a racemase acting on acidic amino acids.

A cyanobacterial bloom occurring in 1998 in lake Tres Pascualas (Concepcion/Chile) was found to be dominated by Microcystis sp. The bloom contained both non-toxic (cyanopeptolin-type) and hepatotoxic (microcystin-type) peptides. Cyanopeptolin structure of the non-toxic peptides (called cyanopeptolin VW-1 and VW-2, respectively) was revealed by matrix assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS) of whole cells, showing dominant molecular ions at m/z = 975 and m/z 995, respectively. On post source decay (PSD), both cyanopeptolins showed fragments deriving from Ahp-Phe-MTyr (3-amino-6-hydroxy-2-piperidone), the characteristic partial structure of cyanopeptolins. The amounts of each of the two cyanopeptolins could only roughly be estimated to be >0.1% of bloom material dry weight. In addition the blooms contained microcystins (20 microg/g bloom dry weight as determined by RP-HPLC, 13 microg/g according to ELISA determination). MALDI-TOF-MS revealed several structural variants of microcystin: MCYST-RR (microcystin with Arg and Arg, indicated by m/z 1,038 and confirmed by PSD revealing a m/z = 135 fragment deriving from the Adda side chain, MCYST-FR (microcystin with Phe and Arg, indicated by m/z = 1,015). The presence of [Asp(3)]-MCYST-LR (microcystin with Leu and Arg, Asp non-methylated, indicated by m/z 981), and [Asp(3)]-MCYST-YR (microcystin with Tyr and Arg, Asp non-methylated, indicated by m/z 1,031) were likely. The relative amounts of the peptides varied between February, April, and May. Whole cell extracts from the bloom material revealed specific enzyme inhibitory activities. The serin-proteases trypsin, plasmin, elastase were inhibited, assumable due to the cyanopeptolins found. Elastase and the cysteine-protease papain were not inhibited, inhibitions of protein kinase and glutathione S-transferase (GST) were low. Strong inhibition was observed with protein-phosphatase-1, likely due to the microcystins present in the samples.

Toxic water blooms from cyanobacteria in lakes and rivers are a worldwide phenomenon. A new technique is presented for the rapid detection of toxic and nontoxic blooms. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was employed to identify mainly peptide metabolites (microcystins, anabaenopeptins, cyanopeptolins, microviridins, microginins and aeruginosins) from microgram quantities of prepared cells within minutes. The spectra show the presence of peptides in strains or water blooms simultaneously. A new compound has been identified using the post source decay (PSD) and collision induced dissociation (CID) mode. This new compound has been defined as anabaenoeptin G. The potential of the method for screening of various secondary metabolite producers for defined products including antibiotics is discussed. Copyright © 1999 John Wiley & Sons, Ltd.

Toxic cyanobacterial blooms are a threat because of secondary metabolite production. We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to identify intact microorganisms. Microgram quantities of prepared cells, including solvent (acetonitrile and ethanol) and alpha-cyano-4-hydroxycinnamic acid matrix, display spectra showing predominantly the secondary metabolites including known microcystins, micropeptin, and anabaenopeptolin. A new cyclic anabaenopeptolin has been identified using the Post-Source-Decay mode. Strains of various origins can easily be typed according to their cyclic peptide production, and toxic and nontoxic algal blooms can be differentiated within minutes.

Ute Matern*, Lukas Oberer, Rocco A. Falchetto, Marcel Erhard, Wilfried A. Konig, Michael Herdman, Jurgen Weckesser Institut fur Biologie II (Mikrobiologie), Albert-Ludwigs-Universitat, Schanzlestrase 1, D-79104 Freiburg i. Br., Germany Novartis Pharma AG, Preclinical Research, CH-4002 Basel, Switzerland AnagnosTec GmbH, Im Biotechnologiepark, D-14943 Luckenwalde, Germany Institut fur Organische Chemie, Universitat Hamburg, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany Unite de Physiologie Microbienne (CNRS URA 2172), Departement de Biochimie et Genetique Moleculaire, Institut Pasteur, 28 Rue du Dr. Roux, F-75724 Paris Cedex 15, France