Dénombrements directs des bactéries des milieux aquatiques par microscopie en épifluorescence : comparaison entre un système d'analyse d'images automatisé (Mudicam®) et l'observation visuelle

La technique de comptage par microscopie en épitluorescence est la méthode la plus performante pour dénombrer la totalité des bactéries présentes dans les milieux aquatiques. Cependant cette technique est longue, fastidieuse et subjective. Afin d'automatiser et de rendre objectif le dénombrement, le microscope à épifluorescence est couplé à un analyseur d'images. Si les systèmes d'analyse d'images sont utilisés pour les mesures de taille des bactéries aquatiques, très peu d'études font état de comparaison entre les dénombrements par analyse d'image et ceux réalisés de façon traditionnelle. Cet article présente les résultats des dénombrements de souches bactériennes de référence et de bactéries des milieux aquatiques, par la technique de microscopie en épifluorescence des cellules bactériennes marquées au DAPI, réalisés simultanément par observation microscopique visuelle (visuel) et par analyse d'images automatisée (automatique).Le système d'analyse d'images est composé d'une caméra vidéo (Lhesa LH40036) de sensibilité de 510-4 lux, d'une carte de numérisation (512 x 512 pixels, 8 bits, cyclope v 2.32, Digital vision) d'un micro-ordinateur 80-386 et d'un logiciel de dénombrement (Mudicam®. EAU). Le système est couplé à un microscope en épilluorescence Olympus BH2.Les dénombrements ont été réalisés d'une part sur des suspensions de souches bactériennes de référence (n = 30) à différents états physiologiques et sur des échantillons d'eaux (n = 50) d'origines diverses (fleuve, eaux saumâtre, marine et résiduaire). La comparaison des deux méthodes est réalisée par un modèle de régression linéaire et une analyse de variance. Les tests statistiques associés permettent de conclure à une bonne concordance entre les deux méthodes. A partir de l'ensemble des dénombrements réalisés, 18 d'entre eux pris au hasard ont été dénombrés de façon manuelle par deux opérateurs et par le système d'analyse d'image. Il apparaît que les différences de comptage les plus élevées correspondent aux dénombrements effectués par chacun des deux opérateurs. Ceci met en évidence que non seulement le système d'analyse d'image permet une quantification rapide des abondances bactériennes, mais en outre il supprime la subjectivité de l'opérateur tout en réalisant des dénombrements aussi précis.Direct counting by epifluorescence microscopy is the best method available to determine total counts of aquatic bacteria. However, microscopic observation is tedious and time-consuming. A more rapid and certainly less subjective way of counting bacteria is to combine epifluorescence microscopy with an image analysis system. Surprisingly, although image analysis is now a relatively common method to measure the size of aquatic bacteria, very few studies have been devoted to the validation of total counts by image-analysis systems. In this paper, we present data on simultaneous determination of total counts of 4'6-diamidino-2-phenylindole (DAPI) stained bacteria by visual means and by image-analysed (Mudicam® system) epifluorescence microscopy methods.The Olympus microscope BH2 is equipped for epifluorescence with a 100 W Hg lamp and a 100x oil immersion objective (Apo UVFL 160/1.3). The image analysis system consists et a high performance (5 x 10-4 lux) video camera (Lhesa LH40036) and an image processor which digitalizes the video image in a grey scale extending from 0 (black) to 255 (white) into a binary image with 512 x 512 pixels (8 bit, cyclope v 2.32, Digital Vision), and image analysis software (MUDICAM®. EAU). The samples were stained with DAPI (final concentration 2.5 µg/ml) and filtered through polycarbonate inters (0.22µm, Nuclepore Corporation). The surface area of the video image is 76 x 111 µm2.The analysed samples come from culture collections of different bacterial strains (n = 30) submitted to different conditions and incubation times to obtain various physiological states (Table 1). The nature water samples were collected from several aquatic ecosystems : Rhône river, Mediterranean sea, Thau lagoon and Montpellier sewage waters (n = 50). The bacterial abundances ranged from 105 to 108 cells/ml and the size range of the cells varied from 0.63 to 17 µm2. Comparisons between the image analysis and visual counts were made on the basic of thirty fields per filter. The image analysis counts are based on a two step procedure. The video image of each microscopie field is first numerised and stored on a hard disk (153 Mo). When all the fields have been stored, the digitized images are submitted to an automatic thresholding which allows background substraction. Automatic counting of bacterial cells is then performed on the basis of object specifications defined by the operator. These specifications concern the minima and maxima values of the area (expressed in pixel numbers) and the fluorescence (expressed in gray levels) of the objects. The MUDICAM®EAU software also provides the mean number of cells per millilitre and the associated variance.Average concentrations and confidence limits are shown in Table 2 for bacterial collection strain cultures and in Table 3 for water samples. When we compared visual and image analysis counts by- linear regression, the ability of the image analysis system to enumerate bacterial cells was clearly demonstrated. With bacterial culture (Fig. 2) and with water samples (Fig. 3), the coefficients of correlation were respectively r = 0.997 and r = 0.996 (p = 0.0001). The slopes of the models are not significantly different from unity and the Y-intercepts are not different from zero. Moreover we have compared the total visual counts of two experimenters and the image-analysed counts on eighteen random samples (Table 4). The variance analysis shows that there is no difference between the three methods, with mean value of 6.09, 6.08 and 6.11 for the image-analysed method, experimenter n° 1 and experimenter n° 2, respectively. While non significant, the greatest difference in counts was obtained between the two experimenters.If may be concluded that the image analyser tested for total counts by epifluorescence microscopy is a precise and rapid procedure for the determination of total bacterial counts. This method may be standardized and its automation allows the analysis of many samples, an important advantage in ecological studies. Storage of the samples also allows one to treat a posteriori some complementary aspects of the total count, such as the double staining of bacteria. The image analyser tested is appropriate for bacterial ecology studies which require epifluorescence microscopy

Utilisation du bouillon sélénite F modifié pour dénombrer Salmonella dans les milieux aquatiques

Ce travail a pour objet de présenter une nouvelle méthode de dénombrement des Salmonella dans différents types d'eaux (de rivière, saumâtre, eaux usées brutes et épurées) basée sur l'utilisation d'un milieu d'enrichissement au sélénite additionné de Novobiocine et de Pril et sur une adaptation de la technique du N.P.P. à l'ensemencement d'échantillons de grands volumes après filtration permettant de quantifier les très faibles concentrations de Salmonella. Les vérifications des performances de cette méthode d'isolement et de quantification sont basées sur l'étude des croissances de différentes souches de Salmonella et d'autres espèces bactériennes dans le milieu d'enrichissement modifié, ainsi que sur les résultats quantitatifs et qualitatifs fournis par cette méthode lorsqu'elle est appliquée à des échantillons d'eau en provenance de l'environnement aquatique. Ces résultats montrent notamment que la méthode proposée est suffisamment sensible pour détecter 1 à 2 Salmonella dans 10 litres d'eau analysée et qu'elle ne parait pas exclure de sérotypes, du moins parmi ceux les plus fréquemment isolés en France. L'efficacité de la méthode standardisée API Z pour l'identification enzymatique du genre Salmonella a été également testée en référence aux résultats de la sérotypie.This paper presents a new method for enumerating Salmonellae in environmental waters (freshwater, brackishwater, sewage and treated waters) using the F Selenite enrichment broth modified by the addition of Novobiocin and Pril, and an adaptation of the M.P.N. method for the inoculation of large amounts of water after filtration to improve the enumeration of low concentrations of Salmonellae. The verification of the performance of this detection and enumeration method are based on the study of the growth of different Salmonellae species and of others bacterial species in the modified enrichment broth, and on the quantitative and qualitative results obtained by the application of this methodology to aquatic environmental samples. These results show on one hand, that the sensitivity of the proposed method allows to enumerate 1 to 2 Salmonellae in 10 liter samples, and on the other hand that this method to net exclude any serovar from those which are the most frequently isolated in France. The efficiency of the API Z standardized method for the Salmonellae enzymatic identification versus serological identification was also verified

Microbial contamination and management scenarios in a Mediterranean coastal lagoon (Etang de Thau, France): application of a Decision Support System within the Integrated Coastal Zone Management context

1 - In the Thau lagoon (Southern Mediterranean Coast) the main anthropogenic pressure is represented by the urban development in the watershed, whilst oyster and mussel farming represents one of the main economical activities in the region. 2 - During the last decade, the increasing organic loads from watershed and urban settlements in the lagoon surroundings have caused a diffuse contamination by faecal bacteria. Also toxic algal blooms have been occurring, impairing water quality with major impacts on shellfish farming, fishery and bathing. 3 - In this study, indicators and scenarios identified for the lagoon have been integrated in a Decision Support System (DSS) to evaluate the best solutions for reducing pressures and improving both water quality and ecosystem status. 4 - The watershed has been analysed with reference to indicators of pollution sources and transfer rates to the lagoon. In parallel, socio-economic indicators and descriptors of urban growth and development have been assessed. Numerical models have been run in order to simulate the lagoon hydrodynamics in relation to both meteorological factors and watershed runoff. The impact of faecal bacteria contamination has been evaluated in terms of economical losses and social conflicts, arising from the restriction of shellfish farming and marketing during contamination events. Finally, the DSS prototype has been applied to the lagoon in support to management and future planning

Seasonal dynamics of active SAR11 ecotypes in the oligotrophic Northwest Mediterranean Sea

A seven-year oceanographic time series in NW Mediterranean surface waters was combined with pyrosequencing of ribosomal RNA (16S rRNA) and ribosomal RNA gene copies (16S rDNA) to examine the environmental controls on SAR11 ecotype dynamics and potential activity. SAR11 diversity exhibited pronounced seasonal cycles remarkably similar to total bacterial diversity. The timing of diversity maxima was similar across narrow and broad phylogenetic clades and strongly associated with deep winter mixing. Diversity minima were associated with periods of stratification that were low in nutrients and phytoplankton biomass and characterised by intense phosphate limitation (turnover time80%) by SAR11 Ia. A partial least squares (PLS) regression model was developed that could reliably predict sequence abundances of SAR11 ecotypes (Q2=0.70) from measured environmental variables, of which mixed layer depth was quantitatively the most important. Comparison of clade-level SAR11 rRNA:rDNA signals with leucine incorporation enabled us to partially validate the use of these ratios as an in-situ activity measure. However, temporal trends in the activity of SAR11 ecotypes and their relationship to environmental variables were unclear. The strong and predictable temporal patterns observed in SAR11 sequence abundance was not linked to metabolic activity of different ecotypes at the phylogenetic and temporal resolution of our study

Characterization of Coastal Urban Watershed Bacterial Communities Leads to Alternative Community-Based Indicators

BACKGROUND: Microbial communities in aquatic environments are spatially and temporally dynamic due to environmental fluctuations and varied external input sources. A large percentage of the urban watersheds in the United States are affected by fecal pollution, including human pathogens, thus warranting comprehensive monitoring. METHODOLOGY/PRINCIPAL FINDINGS: Using a high-density microarray (PhyloChip), we examined water column bacterial community DNA extracted from two connecting urban watersheds, elucidating variable and stable bacterial subpopulations over a 3-day period and community composition profiles that were distinct to fecal and non-fecal sources. Two approaches were used for indication of fecal influence. The first approach utilized similarity of 503 operational taxonomic units (OTUs) common to all fecal samples analyzed in this study with the watershed samples as an index of fecal pollution. A majority of the 503 OTUs were found in the phyla Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. The second approach incorporated relative richness of 4 bacterial classes (Bacilli, Bacteroidetes, Clostridia and alpha-proteobacteria) found to have the highest variance in fecal and non-fecal samples. The ratio of these 4 classes (BBC:A) from the watershed samples demonstrated a trend where bacterial communities from gut and sewage sources had higher ratios than from sources not impacted by fecal material. This trend was also observed in the 124 bacterial communities from previously published and unpublished sequencing or PhyloChip- analyzed studies. CONCLUSIONS/SIGNIFICANCE: This study provided a detailed characterization of bacterial community variability during dry weather across a 3-day period in two urban watersheds. The comparative analysis of watershed community composition resulted in alternative community-based indicators that could be useful for assessing ecosystem health

Organic matter from Artic sea ice loss alters bacterial community structure and function

Continuing losses of multi-year sea ice (MYI) across the Arctic are resulting in first-year ice (FYI) dominating the Arctic ice pack. Melting FYI provides a strong seasonal pulse of dissolved organic matter (DOM) into surface waters; however, the biological impact of this DOM input is unknown. Here we show that DOM additions cause significant and contrasting changes in under-ice bacterioplankton abundance, production and species composition. Utilization of DOM was influenced by molecular size, with 10-100 kDa and >100 kDa DOM fractions promoting rapid growth of particular taxa, while uptake of sulfur and nitrogen-rich low molecular weight organic compounds shifted bacterial community composition. These results demonstrate the ecological impacts of DOM released from melting FYI, with wideranging consequences for the cycling of organic matter across regions of the Arctic Ocean transitioning from multi-year to seasonal sea ice as the climate continues to warm