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

The neutralizing effect of heparin on blood-derived antimicrobial compounds: impact on antibacterial activity and inflammatory response

Acting through a combination of direct and indirect pathogen clearance mechanisms, blood-derived antimicrobial compounds (AMCs) play a pivotal role in innate immunity, safeguarding the host against invading microorganisms. Besides their antimicrobial activity, some AMCs can neutralize endotoxins, preventing their interaction with immune cells and avoiding an excessive inflammatory response. In this study, we aimed to investigate the influence of unfractionated heparin, a polyanionic drug clinically used as anticoagulant, on the endotoxin-neutralizing and antibacterial activity of blood-derived AMCs. Serum samples from healthy donors were pre-incubated with increasing concentrations of heparin for different time periods and tested against pathogenic bacteria (Acinetobacter baumannii, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus) and endotoxins from E. coli, K. pneumoniae, and P. aeruginosa. Heparin dose-dependently decreased the activity of blood-derived AMCs. Consequently, pre-incubation with heparin led to increased activity of LPS and higher values of the pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6). Accordingly, higher concentrations of A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa were observed as well. These findings underscore the neutralizing effect of unfractionated heparin on blood-derived AMCs in vitro and may lead to alternative affinity techniques for isolating and characterizing novel AMCs with the potential for clinical translation

High genetic diversity ofVibrio choleraein the European lake Neusiedler See is associated with intensive recombination in the reed habitat and the long-distance transfer of strains

Coastal marine Vibrio cholerae populations usually exhibit high genetic diversity. To assess the genetic diversity of abundant V. cholerae non-O1/non-O139 populations in the Central European lake Neusiedler See, we performed a phylogenetic analysis based on recA, toxR, gyrB and pyrH loci sequenced for 472 strains. The strains were isolated from three ecologically different habitats in a lake that is a hot-spot of migrating birds and an important bathing water. We also analyzed 76 environmental and human V. cholerae non-O1/non-O139 isolates from Austria and other European countries and added sequences of seven genome-sequenced strains. Phylogenetic analysis showed that the lake supports a unique endemic diversity of V. cholerae that is particularly rich in the reed stand. Phylogenetic trees revealed that many V. cholerae isolates from European countries were genetically related to the strains present in the lake belonging to statistically supported monophyletic clades. We hypothesize that the observed phenomena can be explained by the high degree of genetic recombination that is particularly intensive in the reed stand, acting along with the long distance transfer of strains most probably via birds and/or humans. Thus, the Neusiedler See may serve as a bioreactor for the appearance of new strains with new (pathogenic) properties.The study was financed by the Austrian Science Fund FWF, project nr P21625-B20. In addition, CP and ISD were partially supported by the Austrian Science Fund FWF, P25745-B20. CA thanks to the Ministerio de Educacion, Cultura y Deporte and FEDER funds for the grant AGL2014-58933-P. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.Peer Reviewe

Automated online monitoring of fecal pollution in water by enzymatic methods

RÉSUMÉ: To facilitate the prompt management of public health risks from water resources, the fluorescence-based detection of the enzymatic activity of β-d-glucuronidase (GLUC) has been suggested as a rapid method to monitor fecal pollution. New technological adaptations enable now its automated, near-real-time measurement in a robust and analytically precise manner. Large data sets of high temporal or spatial resolution have been reported from a variety of freshwater resources, demonstrating the great potential of this automated method. However, the fecal indication capacity of GLUC activity and the potential link to health risk is still unclear, presenting considerable limitations. This review provides a critical evaluation of automated, online GLUC-based methods (and alternatives) and defines open questions to be solved before the method can fully support water management

Identification of hotspots of genotoxicological and faecal pollution along the Danube and Sava rivers – the whole river surveys

The level of genotoxic pollution was assessed along the Danube River and its most significant tributary Sava River by measuring the level of DNA damage in aquatic organisms collected from the selected sites. The Danube River survey was conducted within the Joint Danube Survey 3 project in 2013 on 34 sites along the 2285 rkm using mussels (Unio sp.) and fish (Alburnus alburnus) as bioindicators. The Sava River survey was conducted within the Globaqua project in 2015 at 12 sites along 900 rkm using fish (A. alburnus/Alburnoides bipunctatus) as bioindicators. The level of DNA damage was evaluated by the comet assay in haemocytes of mussels and blood cells of fish. The level of faecal pollution along the rivers was studied in parallel with genotoxicological surveys by using the standard indicators, total coliforms, Escherichia coli and Enterococci. Quantification was performed with Colilert/Enterolert Quanti-Tray 2000 and MPN approach. Quantitative PCR (qPCR)-based assays for analysis of human- or animal-associated genetic Bacteroidetes faecal markers have been used for tracking the source of pollution (microbial source tracking -MST). The human-associated BacHum and HF183II, the ruminantassociated BacR and the pig-associated Pig2Bac fecal markers were selected. The hotspots of faecal pollution were detected at both rivers. Presence of pollution was especially evident in the countries in which the legislation related to wastewater treatment and management is not fully implemented. In the case of the Danube River the most critical section of the river was the Pannonian plain (sector VI) while in the case of the Sava River the most affected section was the lower stretch of the river. The results of MST revealed the presence of human-associated fecal markers BacHum and HF183II in the majority of the analyzed samples. High correlation was observed between the standard fecal indicators and human associated faecal markers. Within the Danube survey, the highest levels of DNA damage were recorded in organisms from the section VI, which is under the impact of untreated wastewater discharges. In 2013 the Sava River was characterized with a lower level of both faecal and genotoxic pollution in comparison with the Danube. Similar observations were found within the Sava River survey in 2015 where the level of DNA damage in fish specimens from Sava was lower in comparison with the samples from the Danube. At both rivers detected genotoxic potential was traceable to the deterioration of quality by communal and industrial wastewaters. Acknowledgements: International Commission for the Protection of the Danube River, EU Seventh Framework Program GLOBAQUA (no. 603629-ENV-2013-6.2.1). Ministry of Education, Science and Technological Development of the Republic of Serbia projects no. 173045 and 173025, the bilateral project Serbia and Austria: SER Ev. No. 451-03-01039/2015-09/33

Spring Water of an Alpine Karst Aquifer Is Dominated by a Taxonomically Stable but Discharge-Responsive Bacterial Community

Alpine karst aquifers are important groundwater resources for the provision of drinking water all around the world. Yet, due to difficult accessibility and long-standing methodological limitations, the microbiology of these systems has long been understudied. The aim of the present study was to investigate the structure and dynamics of bacterial communities in spring water of an alpine limestone karst aquifer (LKAS2) under different hydrological conditions (base vs. event flow). The study was based on high-throughput 16S rRNA gene amplicon sequencing, study design and sample selection were guided by hydrology and pollution microbiology data. Spanning more than 27 months, our analyses revealed a taxonomically highly stable bacterial community, comprising high proportions of yet uncultivated bacteria in the suspended bacterial community fraction. Only the three candidate phyla Parcubacteria (OD1), Gracilibacteria (GN02), Doudnabacteria (SM2F11) together with Proteobacteria and Bacteroidetes contributed between 70.0 and 88.4% of total reads throughout the investigation period. A core-community of 300 OTUs consistently contributed between 37.6 and 56.3% of total reads, further supporting the hypothesis of a high temporal stability in the bacterial community in the spring water. Nonetheless, a detectable response in the bacterial community structure of the spring water was discernible during a high-discharge event. Sequence reads affiliated to the class Flavobacteriia clearly increased from a mean proportion of 2.3% during baseflow to a maximum of 12.7% during the early phase of the studied high-discharge event, suggesting direct impacts from changing hydrological conditions on the bacterial community structure in the spring water. This was further supported by an increase in species richness (Chao1) at higher discharge. The combination of these observations allowed the identification and characterization of three different discharge classes (Q1–Q3). In conclusion, we found a taxonomically stable bacterial community prevailing in spring waters from an alpine karst aquifer over the entire study period of more than 2 years. Clear response to changing discharge conditions could be detected for particular bacterial groups, whereas the most responsive group – bacteria affiliated to the class of Flavobacteriia – might harbor potential as a valuable natural indicator of “system disturbances” in karst aquifers

Wastewater based epidemiology in countries with poor wastewater treatment - SARS-CoV-2 RNA in surface waters

Background Wastewater-based epidemiology surveillance of COVID-19 and other outbreaks in the future is a challenge for developing countries, as the majority of households are not connected to sewerage systems. In December 2019, we have detected SARS-CoV-2 RNA in the Danube River at a site that is severely affected by wastewater of Belgrade. Considering that rivers are much more complex systems in comparison to wastewater, additional efforts are needed to address all the factors which might influence the adoption of WBE as an alternative of targeting raw wastewater. Objectives The major objective of this study was to provide a more detailed insight in the potential of SARS-CoV-2 surveillance in Serbian surface waters under consideration of epidemiological, microbiological, physico-chemical and hydro-morphological parameters for epidemiological purposes. Methodology Water samples were collected at 12 sites at Sava and Danube Rivers in the Belgrade city area during the fourth COVID-19 wave in Serbia that started in late February 2021. SARS-CoV-2 RNA was quantified using RT-qPCR with primer sets targeting nucleocapsid (N1 and N2) and envelope (E) protein genes. Microbiological (standard fecal indicator bacteria and microbial faecal source tracking markers), epidemiological, physico-chemical and hydro-morphological parameters were analysed in parallel. Results Out of 44 samples analyzed, 31 were positive for at least one of the target regions of SARS-CoV-2. The results indicated that surveillance of SARS-CoV-2 RNA in surface waters in context with the large amount of epidemiological and environmental metadata can be used as epidemiological early-warning tool in countries with poor wastewater treatment

Recurrent somatic mutations in POLR2A define a distinct subset of meningiomas

RNA polymerase II mediates the transcription of all protein-coding genes in eukaryotic cells, a process that is fundamental to life. Genomic mutations altering this enzyme have not previously been linked to any pathology in humans, which is a testament to its indispensable role in cell biology. On the basis of a combination of next-generation genomic analyses of 775 meningiomas, we report that recurrent somatic p.Gln403Lys or p.Leu438_His439del mutations in POLR2A, which encodes the catalytic subunit of RNA polymerase II (ref. 1), hijack this essential enzyme and drive neoplasia. POLR2A mutant tumors show dysregulation of key meningeal identity genes including WNT6 and ZIC1/ZIC4. In addition to mutations in POLR2A, NF2, SMARCB1, TRAF7, KLF4, AKT1, PIK3CA, and SMO4 we also report somatic mutations in AKT3, PIK3R1, PRKAR1A, and SUFU in meningiomas. Our results identify a role for essential transcriptional machinery in driving tumorigenesis and define mutually exclusive meningioma subgroups with distinct clinical and pathological features

Speciation, Luminescence, and Alkaline Fluorescence Quenching of 4-(2-methylbutyl)aminodipicolinic acid (H2MEBADPA)

4-(2-Methylbutyl)aminodipicolinic acid (H2MEBADPA) has been synthesized and fully characterized in terms of aqueous phase protonation constants (pKa\u27s) and photophysical measurements. The pKa\u27s were determined by spectrophotometric titrations, utilizing a fully sealed titration system. Photophysical measurements consisted of room temperature fluorescence and frozen solution phosphorescence as well as quantum yield determinations at various pH, which showed that only fully deprotonated MEBADPA2– is appreciably emissive. The fluorescence of MEBADPA2– has been determined to be quenched by hydroxide and methoxide anions, most likely through base-catalyzed excited-state tautomerism or proton transfer. This quenching phenomenon has been quantitatively explored through steady-state and time-resolved fluorescence measurements. Utilizing the determined pKas and quenching constants, the fluorescent intensity of MEBADPA2– has been successfully modeled as a function of pH