Quantification of Vibrio cholerae in Lake Neusiedler See by means of classical and molecular biological methods

Vibrio cholerae ist ein bewegliches, Gram-negatives, heterotrophes Stäbchen, das zur Familie der Vibrionaceae gehört. Neben seiner Rolle als wichtiger pathogener Mikroorganismus, ist es auch Bestandteil der normalen Bakterienflora der aquatischen Umwelt, welche Süß-, Brack- und Salzwasser umschließt. Zusätzlich weist es eine duale Lebensform auf, welche entweder Partikel/Zooplankton assoziiert oder frei lebend (planktonisch) sein kann. Von den 200 momentan bekannten V. cholerae Serogruppen können nur Stämme, die zu den Serogruppen O1 und O139 gehören, Cholera, eine schwere Durchfallerkrankung, auslösen. NonO1/nonO139 V. cholerae sind für weniger schwere Durchfallerkrankungen und Blut-, Wund-, Ohr- und Atemwegsinfektionen verantwortlich. Zwischen 2000 und 2007 hat die Weltgesundheitsorganisation über 249 importierten O1/O139 V. cholerae Fällen, in 14 unterschiedlichen europäischen Ländern, berichtet. Im Zeitraum von 2000 bis 2005 traten 14 Infektionen, die auf nonO1/nonO139 V. Cholerae zurückzuführen sind, in Österreich auf. Fünf dieser Infektionen, welche auch einen Todesfall inkludieren, konnten direkt mit dem Neusiedler See in Verbindung gebracht werden. V. cholerae wird üblicherweise in der Umwelt mittels Kultivierung auf Selektivmedium detektiert. Es ist allgemein bekannt, dass V. cholerae unter gewissen Bedingungen, wie Nährstoffmangel und niedriger Temperatur, in den sogenannten VBNC Status (viable but non culturable) wechseln kann, innerhalb dessen es nicht mehr mittels Kultivierungsmethoden detektierbar ist. In dieser Diplomarbeit wurde V. cholerae im Neusiedler See mittels kultivierungsbasierten und molekular biologischen Methoden (Fluoreszenz In Situ Hybridisierung = FISH) an zwei Probenpunkten (Seemitte und Schilfgürtel) quantifiziert. V. cholerae wurde in drei unterschiedlichen Größenfraktionen (> 12 μm, 3-12 μm und 0,2-3 μm) sowie auf Zooplankton (Copepoda Arctodiaptomus spinosus und Cladocera Diaphanosoma mongolianum) von Februar bis August 2008 detektiert. Dafür wurde ein spezifisches FISH Protokoll für dessen Detektion in Umweltproben entwickelt. Mittels Verknüpfung seiner Konzentration mit einer Vielzahl von Umweltparametern, wurde versucht die bevorzugte Nische von diesem Pathogenen im Neusiedler See aufzufinden. Die Ergebnisse zeigen, dass das ausgewählte FISH Protokoll zur Detektion von Bakterien im Labor und im Freiland geeignet ist, sogar wenn sich die Bakterien im VBNC Status befinden (Februar bis Mitte Mai). Ein großer Nachteil der etablierten FISH Methode war, dass die Gegenwart von hohen Partikelkonzentrationen in der Wassersäule zu Zeiten hoher Windgeschwindigkeit (hauptsächlich in der Seemitte), die Genauigkeit und die Zuverlässigkeit der Methode limitierte, was weiter dazu führte, dass die mögliche Beziehung zwischen V. cholerae Konzentrationen und Umweltvariablen maskiert wurde. Beide, FISH und Kultivierung wiesen die höchsten Zellzahlen im Juli, mit 22.000 – 26.000 CFU L-1 und 45.000 - 46.000 CFU L-1, und abnehmenden Werten an den zwei folgenden Probenpunkten auf, was auf einen Abfall von Partikeln in der Wassersäule, aufgrund milder Wetterbedingungen, zurückzuführen ist. Von Mai bis August wurde mit beiden Methoden ähnliche Resultate erreicht und es gab eine signifikant positive Korrelation zwischen FISH und kultivierungsbasierten V. cholerae Konzentrationen. Es traten signifikant höhere V. cholerae Konzentrationen in der Seemitte als im Schilfgürtel auf. In beiden, der Seemitte und dem Schilfgürtel, wurden mittels Kultivierung die meisten Bakterien in der Größenfraktion 3- 12 μm detektiert, was darauf hinweist, dass diese Partikelfraktion bestimmte Umweltbedingungen aufweist, welche die Kultivierung von V. cholerae verstärkt. Im Gegensatz dazu, traten mittels FISH die meisten Bakterien in der Größenfraktion 0,2-3 μm auf. Da es sich herausgestellt hat, dass die Zuverlässigkeit und Ausbeute an Zellen mittels FISH von der Partikelkonzentration im Wasser abhängt und die Fraktion 0,2-3 μm die partikelärmste Fraktion war, könnte dieses Ergebnis möglicherweise auf eine methodische Verzerrung zurückzuführen sein. Im Gegensatz zum allgemeinen Wissensstand, zeigten die nonO1/nonO139 isolierten V. cholerae Stämme aus dem Neusiedler See eine sehr schwache Assoziation mit Copepoden, aber eine sehr starke Verknüpfung mit der dominierenden Cladoceren Art. Das impliziert, dass es eine spezifische Interaktion zwischen bestimmten Zooplankton Arten und V. cholerae Arten geben muss. Außerdem zeigt der große Unterschied an Cladoceren assoziierten V. cholerae Konzentrationen zwischen der FISH und kultivierungsbasierten Methode, dass die meisten Zooplankton angehefteten Bakterien die inneren Oberflächen kolonisieren und nicht auf dem Exoskelett vorkommen. Zusammenfassend ist zu sagen, dass V. cholerae Stämme im Neusiedler See einige Überlebensstrategien (Anheftung an Cladoceren, Assoziation mit der 3-12 μm Partikelfraktion, planktonisches Wachstum) zu haben scheinen und anscheinend nicht nur eine, spezialisierte ökologische Nische besetzten.Vibrio cholerae is a motile, Gram-negative, heterotrophic rod that belongs to the family Vibrionaceae. Besides being an important human pathogen, it is a component of the normal bacterial flora of aquatic environments, including fresh, brackish and sea water. In the environment, at least two modes of life have been discussed, either particle/zooplankton associated or free living (planktonic). Of the 200 currently recognized serogroups of V. cholerae, only strains belonging to serogroups O1 and O139 can cause cholerae, a fatal diarrhoeal disease. NonO1/nonO139 V. cholerae strains are responsible for less severe watery diarrhoe and infections of blood, wound, ear and the respiratory tract. Between 2000 and 2007, the World Health Organization (WHO) reported of 249 imported O1/O139 V. cholerae cases in 14 different European countries. During the period from 2000 to 2005, 13 infections due to environmental nonO1/nonO139 V. cholerae strains were documented in Austria. Five infections thereof could be directly related to Lake Neusiedler See, including one case of death. Vibrio cholerae is usually detected from environmental samples by means of cultivation on selective media. However, under certain circumstances, like nutrient deprivation and low temperature V. cholerae changes to the so-called VBNC status (viable but non cultivable) where it is undetectable by cultivation. In this study, environmental V. cholerae from the lake Neusiedler See were quantified by a cultivation based and a molecular biological method (fluorescence in situ hybridisation = FISH) at two different sampling sites (lake center and reed belt). V.cholerae were detected in three different size fractions (> 12 µm, 3-12 µm and 0.2-3 µm) as well as on zooplankton (copepods Arctodiaptomus spinosus and the cladocerans Diaphanosoma mongolianum) from February till August, 2008. Therefore a specific FISH protocol was developed for its detection in environmental samples. By relating its abundance to a variety of environmental parameters it was attempted to trace the preferred ecological niche of this human pathogen in the Lake Neusiedler See. The results indicate that the selected FISH protocol was suitable for detection of the bacteria in laboratory experiments and environmental samples even when the cells were in the VBNC status (February to mid May). However, one major disadvantage of the established FISH protocol was that the presence of high particle concentrations in the water column at periods of high wind speed (mainly in the lake center) limited the accuracy and reliability of the method which further led to a masking of possible relationships of V. cholerae concentrations to environmental variables. Both, FISH and cultivation showed cell number peaks in July with 22.000 - 26.000 CFU L-1 and 45.000 - 46.000 CFU L-1, and decreasing values on the two following sampling dates, which could be attributed to a decrease in particles in the water column due to calm weather situations. From May to August, both methods yielded similar results and there was a significant positive correlation between the FISH and cultivation based V. cholerae concentrations. Significantly higher V. cholerae concentrations were present in the lake center than in the reed belt. In both, the lake center and the reed belt most of the cells could be detected in the size fraction 3-12 μm by cultivation indicating that this particle fraction contains certain environmental features that enhance V. cholerae cultivability. In contrast, by means of FISH most V. cholerae cells occurred in the size fraction 0.2-3 μm. Because the reliability and recovery of cells by the FISH method proved to be dependent on the particle concentration in the water and the 0.2-3 μm size fraction was the poorest in particles this may be due to a methodical bias. Surprisingly, in contrast to common knowledge, the nonO1/nonO139 V. cholerae strains isolated from the lake Neusiedler See showed very weak association with copepods but a strong association with the dominant cladocerans species. This implicates that there is a specific interaction between certain zooplankton species and specific V. cholerae strains. Moreover, the large difference in cladoceran associated V. cholerae numbers between the FISH method and the cultivation based approach indicates that most of the zooplankton attached cells colonize inner surfaces and are not present on the exoskeleton. Summing up, the V. cholerae strains in Lake Neusiedler See seem to have several life strategies (attachment to cladocerans, association with the 3 – 12 µm particle fraction, planktonic growth) and do not occupy a single specialized ecological niche

Interaction of Vibrio cholerae non-O1/non-O139 with Copepods, Cladocerans and Competing Bacteria in the Large Alkaline Lake Neusiedler See, Austria

Vibrio cholerae is a human pathogen and natural inhabitant of aquatic environments. Serogroups O1/O139 have been associated with epidemic cholera, while non-O1/non-O139 serogroups usually cause human disease other than classical cholera. V. cholerae non-O1/non-O139 from the Neusiedler See, a large Central European lake, have caused ear and wound infections, including one case of fatal septicaemia. Recent investigations demonstrated rapid planktonic growth of V. cholerae non-O1/non-O139 and correlation with zooplankton biomass. The aim of this study was to elucidate the interaction of autochthonous V. cholerae with two dominant crustacean zooplankton species in the lake and investigate the influence of the natural bacterial community on this interaction. An existing data set was evaluated for statistical relationships between zooplankton species and V. cholerae and co-culture experiments were performed in the laboratory. A new fluorescence in situ hybridisation protocol was applied for quantification of V. cholerae non-O1/non-O139 cells, which significantly reduced analysis time. The experiments clearly demonstrated a significant relationship of autochthonous V. cholerae non-O1/non-O139 with cladocerans by promoting growth of V. cholerae non-O1/non-O139 in the water and on the surfaces of the cladocerans. In contrast, copepods had a negative effect on the growth of V. cholerae non-O1/non-O139 via competing bacteria from their surfaces. Thus, beside other known factors, biofilm formation by V. cholerae on crustacean zooplankton appears to be zooplankton taxon specific and may be controlled by the natural bacterial community. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00248-010-9764-9) contains supplementary material, which is available to authorized users

Compound-specific δ¹³C analyses reveal sterol metabolic constraints in an aquatic invertebrate

RATIONALE: Dietary sterol deficiencies can have severe life history consequences for consumers. Compound-specific stable isotope analysis (CSIA) was applied to the exploration of the sterol metabolic constraints and bioconversion capacities of the amphipod Gammarus roeselii. Evaluating structural sterol requirements has great potential to improve our understanding of the ecological relevance of sterols as limiting nutrients.METHODS: Juvenile G. roeselii were reared on food mixtures consisting of different ratios of the two algae Scenedesmus obliquus (cultivated with 13C-labeled NaHCO3) and Nannochloropsis limnetica (unlabeled), which have been shown previously to differ in food quality. We measured the sterol content and composition using a gas chromatograph equipped with a flame ionization detector and the δ13C values of sterols using compound-specific isotope ratio mass spectrometry to examine potential sterol-mediated nutritional constraints of G. roeselii.RESULTS: In the food mixtures, δ13C values of cholesterol, synthesized by N. limnetica, were 25‰and those of the Δ7-phytosterols, chondrillasterol and fungisterol, synthesized by S. obliquus, were 7 and 18‰, respectively. Although the cholesterol concentrations in G. roeselii decreased with increasing proportion of dietary S. obliquus, the δ13C values remained constant at 25‰. Lathosterol, which appeared in G. roeselii at high dietary proportions of S. obliquus, had a δ13C value of 35‰.CONCLUSIONS: We provide evidence that the the Δ7-phytosterols present in S. obliquus cannot be metabolized to cholesterol in G. roeselii, resulting in the accumulation of lathosterol in the animals and potentially in sterol-limited growth. These findings emphasize the advantage of CSIA in revealing the physiological mechanisms associated with nutritional constraints

Spectrum of novel mutations found in Waardenburg syndrome types 1 and 2: implications for molecular genetic diagnostics

Objectives: Till date, mutations in the genes PAX3 and MITF have been described in Waardenburg syndrome (WS), which is clinically characterised by congenital hearing loss and pigmentation anomalies. Our study intended to determine the frequency of mutations and deletions in these genes, to assess the clinical phenotype in detail and to identify rational priorities for molecular genetic diagnostics procedures. Design: Prospective analysis. Patients: 19 Caucasian patients with typical features of WS underwent stepwise investigation of PAX3 and MITF. When point mutations and small insertions/deletions were excluded by direct sequencing, copy number analysis by multiplex ligation-dependent probe amplification was performed to detect larger deletions and duplications. Clinical data and photographs were collected to facilitate genotype-phenotype analyses. Setting: All analyses were performed in a large German laboratory specialised in genetic diagnostics. Results: 15 novel and 4 previously published heterozygous mutations in PAX3 and MITF were identified. Of these, six were large deletions or duplications that were only detectable by copy number analysis. All patients with PAX3 mutations had typical phenotype of WS with dystopia canthorum (WS1), whereas patients with MITF gene mutations presented without dystopia canthorum (WS2). In addition, one patient with bilateral hearing loss and blue eyes with iris stroma dysplasia had a de novo missense mutation (p. Arg217Ile) in MITF. MITF 3-bp deletions at amino acid position 217 have previously been described in patients with Tietz syndrome (TS), a clinical entity with hearing loss and generalised hypopigmentation. Conclusions: On the basis of these findings, we conclude that sequencing and copy number analysis of both PAX3 and MITF have to be recommended in the routine molecular diagnostic setting for patients, WS1 and WS2. Furthermore, our genotype-phenotype analyses indicate that WS2 and TS correspond to a clinical spectrum that is influenced by MITF mutation type and position

Comparative Analysis of Platelet-Derived Extracellular Vesicles Using Flow Cytometry and Nanoparticle Tracking Analysis

Growing interest in extracellular vesicles (EVs) has prompted the advancements of protocols for improved EV characterization. As a high-throughput, multi-parameter, and single particle technique, flow cytometry is widely used for EV characterization. The comparison of data on EV concentration, however, is hindered by the lack of standardization between different protocols and instruments. Here, we quantified EV counts of platelet-derived EVs, using two flow cytometers (Gallios and CytoFLEX LX) and nanoparticle tracking analysis (NTA). Phosphatidylserine-exposing EVs were identified by labelling with lactadherin (LA). Calibration with silica-based fluorescent beads showed detection limits of 300 nm and 150 nm for Gallios and CytoFLEX LX, respectively. Accordingly, CytoFLEX LX yielded 40-fold higher EV counts and 13-fold higher counts of LA+CD41+ EVs compared to Gallios. NTA in fluorescence mode (F-NTA) demonstrated that only 9.5% of all vesicles detected in scatter mode exposed phosphatidylserine, resulting in good agreement of LA+ EVs for CytoFLEX LX and F-NTA. Since certain functional characteristics, such as the exposure of pro-coagulant phosphatidylserine, are not equally displayed across the entire EV size range, our study highlights the necessity of indicating the size range of EVs detected with a given approach along with the EV concentration to support the comparability between different studies

The capacity of the TNF family members 4-1BBL, OX40L, CD70, GITRL, CD30L and LIGHT to costimulate human T cells

Activating signals generated by members of the tumour necrosis factor receptor superfamily upon interaction with their cognate ligands play important roles in T-cell responses. Members of the tumour necrosis factor family namely 4-1BBL, OX40L, CD70, GITRL, LIGHT and CD30L have been described to function as costimulatory molecules by binding such receptors on T cells. Using our recently described system of T-cell stimulator cells we have performed the first study where all these molecules have been assessed and compared regarding their capacity to costimulate proliferation and cytokine production of human T cells. 4-1BBL, which we found to be the most potent molecule in this group, was able to mediate sustained activation and proliferation of human T cells. OX40L and CD70 were also strong inducers of T-cell proliferation, whereas the costimulatory capacity of human GITRL was significantly lower. Importantly CD30L and LIGHT consistently failed to act costimulatory on human T cells, and we therefore suggest that these molecules might be functionally distinct from the costimulatory members of this family