Microarray (phylochip) analysis of freshwater pathogens at several sites along the Northern German coast transecting both estuarine and freshwaters.

Monitoring the quality of drinking water is an important issue for public health. Two of the main objectives of the European Project µAQUA were (i) the development of specific probes to detect and quantify pathogens in drinking water and (ii) the design of standardized sampling programs of water from different sources in Europe in order to obtain sufficient material for downstream analysis. Our phylochip contains barcodes that specifically identify freshwater pathogens for enabling the detection of organisms that can be risks for human health. Monitoring for organisms with molecular tools is rapid, more accurate and more reliable than traditional methods. Rapid detection means that mitigation strategies come into play faster with less harm to the community and to humans. Samples were collected from several waters in France, Germany, Ireland, Italy and Turkey over 2 years. We present microarray results for the presence of freshwater pathogens from brackish and freshwater sites in Northern Germany, and cyanobacterial cell numbers inferred from these sites. In a companion study from the same samples, cyanobacterial toxins were analyzed using two methods and those sites with highest toxin values also had highest cell numbers as inferred from this microarray study.

Characterization of N-Acyl Homoserine Lactones in Vibrio tasmaniensis LGP32 by a Biosensor-Based UHPLC-HRMS/MS Method.

Since the discovery of quorum sensing (QS) in the 1970s, many studies have demonstrated that Vibrio species coordinate activities such as biofilm formation, virulence, pathogenesis, and bioluminescence, through a large group of molecules called N-acyl homoserine lactones (AHLs). However, despite the extensive knowledge on the involved molecules and the biological processes controlled by QS in a few selected Vibrio strains, less is known about the overall diversity of AHLs produced by a broader range of environmental strains. To investigate the prevalence of QS capability of Vibrio environmental strains we analyzed 87 Vibrio spp. strains from the Banyuls Bacterial Culture Collection (WDCM911) for their ability to produce AHLs. This screening was based on three biosensors, which cover a large spectrum of AHLs, and revealed that only 9% of the screened isolates produced AHLs in the defined experimental conditions. Among these AHL-producing strains, Vibrio tasmaniensis LGP32 is a well-known pathogen of bivalves. We further analyzed the diversity of AHLs produced by this strain using a sensitive bioguided UHPLC-HRMS/MS approach (Ultra-High-Performance Liquid Chromatography followed by High-Resolution tandem Mass Spectrometry) and we identified C10-HSL, OH-C12-HSL, oxo-C12-HSL and C14:1-HSL as QS molecules. This is the first report that documents the production of AHL by Vibrio tasmaniensis LGP32.

Seasonal dynamics of freshwater pathogens as measured by microarray at Lake Sapanca, a drinking water source in the north-eastern part of Turkey.

Monitoring drinking water quality is an important public health issue. Two objectives from the 4 years, six nations, EU Project µAqua were to develop hierarchically specific probes to detect and quantify pathogens in drinking water using a PCR-free microarray platform and to design a standardised water sampling program from different sources in Europe to obtain sufficient material for downstream analysis. Our phylochip contains barcodes (probes) that specifically identify freshwater pathogens that are human health risks in a taxonomic hierarchical fashion such that if species is present, the entire taxonomic hierarchy (genus, family, order, phylum, kingdom) leading to it must also be present, which avoids false positives. Molecular tools are more rapid, accurate and reliable than traditional methods, which means faster mitigation strategies with less harm to humans and the community. We present microarray results for the presence of freshwater pathogens from a Turkish lake used drinking water and inferred cyanobacterial cell equivalents from samples concentrated from 40 into 1 L in 45 min using hollow fibre filters. In two companion studies from the same samples, cyanobacterial toxins were analysed using chemical methods and those dates with highest toxin values also had highest cell equivalents as inferred from this microarray study.

Dynamics of Vibrio cholerae abundance in Austrian saline lakes, assessed with quantitative solid-phase cytometry.

In order to elucidate the main predictors of Vibrio cholerae dynamics and to estimate the risk of Vibrio cholera-related diseases, a recently developed direct detection approach based on fluorescence in situ hybridization and solid-phase cytometry (CARD-FISH/SPC) was applied in comparison to cultivation for water samples from the lake Neusiedler See, Austria and three shallow alkaline lakes over a period of 20 months. Vibrio cholerae attached to crustacean zooplankton was quantified via FISH and epifluorescence microscopy. Concentrations obtained by CARD-FISH/SPC were significantly higher than those obtained by culture in 2011, but were mostly of similar magnitude in 2012. Maximum cell numbers were 1.26 × 10(6) V. cholerae per L in Neusiedler See and 7.59 × 10(7) V. cholerae per L in the shallow alkaline lakes. Only on a few occasions during summer was the crustacean zooplankton the preferred habitat for V. cholerae. In winter, V. cholerae was not culturable but could be quantified at all sites with CARD-FISH/SPC. Beside temperature, suspended solids, zooplankton and ammonium were the main predictors of V. cholerae abundance in Neusiedler See, while in the shallow alkaline lakes it was organic carbon, conductivity and phosphorus. Based on the obtained concentrations a first estimation of the health risk for visitors of the lake could be performed.

[Sources and fate of pathogenic microorganisms in aquatic environments]. / Sources et devenir des micro-organismes pathogènes dans les environnements aquatiques.

A large number of human waterborne infections are caused by microorganisms including viruses, bacteria or protozoa. These microorganisms are either naturally presents in aquatic environments, or come from fecal sources. They may stay in aquatic ecosystems for a long time before contaminating a new host. Aquatic environments are subjected to high variability of physico- chemical parameters. They include a microbial community that is more or less adapted to these environmental changes. Thus, numerous interactions occur between microorganisms, which are pathogenic or not, and most of them are still unknown. This review focused on interactions between pathogen microorganisms and their environment. More specifically we addressed the question of the diversity of the sources of contamination, their contribution to the microbiological pollution of the aquatic environment, and the ability of some pathogens to survive adverse environmental conditions.

Genomic diversity and metabolic potential of marine Pseudomonadaceae.

Recent changes in the taxonomy of the Pseudomonadaceae family have led to the delineation of three new genera (Atopomonas, Halopseudomonas and Stutzerimonas). However, the genus Pseudomonas remains the most densely populated and displays a broad genetic diversity. Pseudomonas are able to produce a wide variety of secondary metabolites which drives important ecological functions and have a great impact in sustaining their lifestyles. While soilborne Pseudomonas are constantly examined, we currently lack studies aiming to explore the genetic diversity and metabolic potential of marine Pseudomonas spp. In this study, 23 Pseudomonas strains were co-isolated with Vibrio strains from three marine microalgal cultures and rpoD-based phylogeny allowed their assignment to the Pseudomonas oleovorans group (Pseudomonas chengduensis, Pseudomonas toyotomiensis and one new species). We combined whole genome sequencing on three selected strains with an inventory of marine Pseudomonas genomes to assess their phylogenetic assignations and explore their metabolic potential. Our results revealed that most strains are incorrectly assigned at the species level and half of them do not belong to the genus Pseudomonas but instead to the genera Halopseudomonas or Stutzerimonas. We highlight the presence of 26 new species (Halopseudomonas (n = 5), Stutzerimonas (n = 7) and Pseudomonas (n = 14)) and describe one new species, Pseudomonas chaetocerotis sp. nov. (type strain 536T = LMG 31766T = DSM 111343T). We used genome mining to identify numerous BGCs coding for the production of diverse known metabolites (i.e., osmoprotectants, photoprotectants, quorum sensing molecules, siderophores, cyclic lipopeptides) but also unknown metabolites (e.g., ARE, hybrid ARE-DAR, siderophores, orphan NRPS gene clusters) awaiting chemical characterization. Finally, this study underlines that marine environments host a huge diversity of Pseudomonadaceae that can drive the discovery of new secondary metabolites.

Evidence of a Large Diversity of N-acyl-Homoserine Lactones in Symbiotic Vibrio fischeri Strains Associated with the Squid Euprymna scolopes.

Vibrio fischeri possesses a complex AHL-mediated Quorum-sensing (QS) system including two pathways, LuxI/R (3-oxo-C6-HSL and C6-HSL) and AinS/R (C8-HSL), which are important for the regulation of physiological traits. Diverse QS-dependent functional phenotypes have been described in V. fischeri; however, AHL diversity is still underestimated. In the present study, we investigated AHL diversity in five symbiotic V. fischeri strains with distinct phenotypic properties using UHPLC-HRMS/MS. The results obtained (1) revealed an unexpectedly high diversity of signaling molecules, (2) emphasized the complexity of QS in V. fischeri, and (3) highlight the importance of understanding the specificity of AHL-mediated QS.

Genetic diversity and phenotypic plasticity of AHL-mediated Quorum sensing in environmental strains of Vibrio mediterranei.

N-Acyl homoserine lactone (AHL)-mediated Quorum sensing (QS) is one of the most studied social behavior among Proteobacteria. However, despite the current knowledge on QS-associated phenotypes such as bioluminescence, biofilm formation, or pathogenesis, the characterization of environmental factors driving QS in realistic ecological settings remains scarce. We investigated the dynamics of AHL and AHL-producing Vibrio among 840 isolates  collected fortnightly from the Salses-Leucate Mediterranean lagoon in spring and summer 2015 and 2016. Vibrio isolates were characterized by gyrB gene sequencing, Enterobacterial repetitive intergenic consensus polymerase chain reaction, and genome sequencing, and AHL production was investigated by a biosensors-based UHPLC-HRMS/MS approach. Our results revealed, for the first time, a succession of V. mediterranei isolates with different AHL production phenotypes over time and this dynamics was observed in a single genotype (average genomic nucleotide identity >99.9). A multivariate DistLM analysis revealed that 83.4% of the temporal variation of V. mediterranei QS phenotypes was explained by environmental variables. Overall, our results suggest that isolates of a single genotype are able to change their QS phenotypes in response to environmental conditions, highlighting the phenotypic plasticity of bacterial communication in the environment.

Biosensing platforms for Vibrio bacteria detection based on whole cell and nucleic acid analysis: A review.

Vibrio related illnesses are increasing worldwide in humans and marine animals. The detection of these bacteria is still mainly performed using traditional microbiological methods based on culture grown on differential agar media which are labor intensive, time consuming and unsuitable for in-field and high-throughput analysis. To overcome these limitations, biosensing platforms have emerged as promising alternative tools for rapid, sensitive, and real-time detection of Vibrio species in clinical, food and environmental samples. In this review, we will focus on strip test devices, and on optical and electrochemical biosensors developed for Vibrio analysis. Particular attention is given to sample preparation techniques.

Spatiotemporal Dynamics of Total Viable Vibrio spp. in a NW Mediterranean Coastal Area.

A cellular approach combining Direct Viable Counting and Fluorescent In Situ Hybridization using a one-step multiple-probe technique and Solid Phase Cytometry (DVC-FISH-SPC) was developed to monitor total viable vibrios and cover the detection of a large diversity of vibrios. FISH combined three probes in the same assay and targeted sequences located at different positions on the 16S rRNA of Vibrio and Aliivibrio members. We performed a 10-month in situ study to investigate the weekly dynamics of viable vibrios relative to culturable counts at two northwestern Mediterranean coastal sites, and identified the key physicochemical factors for their occurrence in water using a multivariate analysis. Total viable and culturable cell counts showed the same temporal pattern during the warmer season, whereas the ratios between both methods were inverted during the colder seasons (<15°C), indicating that some of the vibrio community had entered into a viable but non-culturable (VBNC) state. We confirmed that Seawater Surface Temperature explained 51-62% of the total variance in culturable counts, and also showed that the occurrence of viable vibrios is controlled by two variables, pheopigment (15%) and phosphate (12%) concentrations, suggesting that other unidentified factors play a role in maintaining viability.

Monitoring of freshwater toxins in European environmental waters by using novel multi-detection methods.

Monitoring the quality of freshwater is an important issue for public health. In the context of the European project µAqua, 150 samples were collected from several waters in France, Germany, Ireland, Italy, and Turkey for 2 yr. These samples were analyzed using 2 multitoxin detection methods previously developed: a microsphere-based method coupled to flow-cytometry, and an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. The presence of microcystins, nodularin, domoic acid, cylindrospermopsin, and several analogues of anatoxin-a (ATX-a) was monitored. No traces of cylindrospermopsin or domoic acid were found in any of the environmental samples. Microcystin-LR and microcystin-RR were detected in 2 samples from Turkey and Germany. In the case of ATX-a derivatives, 75% of samples contained mainly H2 -ATX-a and small amounts of H2 -homoanatoxin-a, whereas ATX-a and homoanatoxin-a were found in only 1 sample. These results confirm the presence and wide distribution of dihydro derivatives of ATX-a toxins in European freshwaters. Environ Toxicol Chem 2017;36:645-654. © 2016 SETAC.

Assessment of a new technique combining a viability test, whole-cell hybridization and laser-scanning cytometry for the direct counting of viable Enterobacteriaceae cells in drinking water.

A new direct approach, called direct viable count (DVC)-FISH-ScanRDI, combining viability measurement, specific detection and sensitive enumeration of highly diluted Enterobacteriaceae cells, was assessed during the summer in water samples from a North American drinking water treatment plant and its distribution system. Major results of this field investigation show a higher sensitivity of the DVC-FISH-ScanRDI approach in enumerating viable Enterobacteriaceae cells in distributed drinking water, relative to a culture-based method, and the increased concentration of viable but non-culturable (VBNC) Enterobacteriaceae cells in distributed water for temperatures above 18 degrees C.

Detection and enumeration of coliforms in drinking water: current methods and emerging approaches.

The coliform group has been used extensively as an indicator of water quality and has historically led to the public health protection concept. The aim of this review is to examine methods currently in use or which can be proposed for the monitoring of coliforms in drinking water. Actually, the need for more rapid, sensitive and specific tests is essential in the water industry. Routine and widely accepted techniques are discussed, as are methods which have emerged from recent research developments.Approved traditional methods for coliform detection include the multiple-tube fermentation (MTF) technique and the membrane filter (MF) technique using different specific media and incubation conditions. These methods have limitations, however, such as duration of incubation, antagonistic organism interference, lack of specificity and poor detection of slow-growing or viable but non-culturable (VBNC) microorganisms. Nowadays, the simple and inexpensive membrane filter technique is the most widely used method for routine enumeration of coliforms in drinking water.The detection of coliforms based on specific enzymatic activity has improved the sensitivity of these methods. The enzymes beta-D galactosidase and beta-D glucuronidase are widely used for the detection and enumeration of total coliforms and Escherichia coli, respectively. Many chromogenic and fluorogenic substrates exist for the specific detection of these enzymatic activities, and various commercial tests based on these substrates are available. Numerous comparisons have shown these tests may be a suitable alternative to the classical techniques. They are, however, more expensive, and the incubation time, even though reduced, remains too long for same-day results. More sophisticated analytical tools such as solid phase cytometry can be employed to decrease the time needed for the detection of bacterial enzymatic activities, with a low detection threshold. Detection of coliforms by molecular methods is also proposed, as these methods allow for very specific and rapid detection without the need for a cultivation step. Three molecular-based methods are evaluated here: the immunological, polymerase chain reaction (PCR) and in-situ hybridization (ISH) techniques. In the immunological approach, various antibodies against coliform bacteria have been produced, but the application of this technique often showed low antibody specificity. PCR can be used to detect coliform bacteria by means of signal amplification: DNA sequence coding for the lacZ gene (beta-galactosidase gene) and the uidA gene (beta-D glucuronidase gene) has been used to detect total coliforms and E. coli, respectively. However, quantification with PCR is still lacking in precision and necessitates extensive laboratory work. The FISH technique involves the use of oligonucleotide probes to detect complementary sequences inside specific cells. Oligonucleotide probes designed specifically for regions of the 16S RNA molecules of Enterobacteriaceae can be used for microbiological quality control of drinking water samples. FISH should be an interesting viable alternative to the conventional culture methods for the detection of coliforms in drinking water, as it provides quantitative data in a fairly short period of time (6 to 8 h), but still requires research effort. This review shows that even though many innovative bacterial detection methods have been developed, few have the potential for becoming a standardized method for the detection of coliforms in drinking water samples.

Colorimetric and electrochemical genosensors for the detection of Escherichia coli DNA without amplification in seawater.

Monitoring seawater, particularly recreational water, for indicator bacteria presence is required to protect the public from exposure to fecal pollution and to guarantee the safety of the swimming areas. Two methods for the detection and quantification of Escherichia coli DNA were developed: a colorimetric assay in a microplate and an electrochemical biosensor. These assays were based on the double hybridization recognition of a single-strand DNA capture probe immobilized onto the microplate or the screen-printed carbon electrode to its complementary ssDNA, which is hybridized with an ssDNA signal probe labeled with horseradish peroxidase enzyme. The hybridization recognition step used the colorimetric monitoring of the oxidation state of the 3,3',5,5'-tetramethylbenzidine. The electrochemical monitoring of the oxidation state of 5 methyl-phenazinium methyl sulfate was allowed when the horseradish-peroxidase was in the presence of the mediator (5 methyl-phenazinium methyl sulfate and hydrogen peroxide). These approaches allow for the detection and quantification of 10(2) to 10(3) cells of E. coli in 5l of seawater samples in less than 5h. Detection was achieved without a nucleic acid amplification step. The specificity of the two methods against E. coli was demonstrated by testing a panel of bacteria. The two methods can be used for on-site monitoring of seawater quality.

Rapid and sensitive enumeration of viable diluted cells of members of the family enterobacteriaceae in freshwater and drinking water.

Water quality assessment involves the specific, sensitive, and rapid detection of bacterial indicators and pathogens in water samples, including viable but nonculturable (VBNC) cells. This work evaluates the specificity and sensitivity of a new method which combines a fluorescent in situ hybridization (FISH) approach with a physiological assay (direct viable count [DVC]) for the direct enumeration, at the single-cell level, of highly diluted viable cells of members of the family Enterobacteriaceae in freshwater and drinking water after membrane filtration. The approach (DVC-FISH) uses a new direct detection device, the laser scanning cytometer (Scan RDI). Combining the DVC-FISH method on a membrane with Scan RDI detection makes it possible to detect as few as one targeted cell in approximately 10(8) nontargeted cells spread over the membrane. The ability of this new approach to detect and enumerate VBNC enterobacterial cells in freshwater and drinking water distribution systems was investigated and is discussed.