Biomedical subjects

P Lebaron

Publications and source records attributed to P Lebaron.

At least 19 recordsLinked to original sources

PubMed2006

Structure of virioplankton in the Charente Estuary (France): transmission electron microscopy versus pulsed field gel electrophoresis.

Changes in the composition of viral communities were investigated along a salinity gradient and at different times by means of transmission electron microscopy (TEM) and pulsed field gel electrophoresis (PFGE). Samples were collected in fresh (Charente River), estuarine (Charente Estuary), and coastal (Pertuis d'Antioche, French Atlantic coast) waters. Both methods revealed similar patterns in viral community structure with a dominance of small viral particles (capsid and genome size). Viruses with a head size below 65 nm made up 71 +/- 5% of total virus-like particles, and virus-like genomes (VLG) below 100 kb accounted for 89 +/- 9% of total VLG. Despite this apparent stability of virioplankton composition over spatial scale (salinity gradient), the occurrence of large viruses (capsid and genome size) in estuarine and seawater samples indicated the presence of viral populations specific to a geographical location. Temporal changes in the structure (capsid and genome size) of viral communities were more pronounced than those reported at the spatial scale. From January to May 2003, seasonal changes in viral abundance and bacterial production occurred concomitantly with an increase in viral genomic diversity (richness), suggesting that virioplankton composition was strongly linked to changes in microbial activity and/or in the structure of the host communities. Although PFGE and TEM yielded complementary results in the description of virioplankton structures, it seems that the use of PFGE alone should be enough for the monitoring of community changes.

Capsid↗

PubMed2006

Rapid quantification of the toxic alga Prymnesium parvum in natural samples by use of a specific monoclonal antibody and solid-phase cytometry.

The increasing incidence of harmful algal blooms around the world and their associated health and economic effects require the development of methods to rapidly and accurately detect and enumerate the target species. Here we describe use of a solid-phase cytometer to detect and enumerate the toxic alga Prymnesium parvum in natural samples, using a specific monoclonal antibody and indirect immunofluorescence. The immunoglobulin G antibody 16E4 exhibited narrow specificity in that it recognized several P. parvum strains and a Prymnesium nemamethecum strain but it did not cross-react with P. parvum strains from Scandinavia or any other algal strains, including species of the closely related genus Chrysochromulina. Prymnesium sp. cells labeled with 16E4 were readily detected by the solid-phase cytometer because of the large fluorescence signal and the signal/noise ratio. Immunofluorescence detection and enumeration of cultured P. parvum cells preserved with different fixatives showed that the highest cell counts were obtained when cells were fixed with either glutaraldehyde or formaldehyde plus the cell protectant Pluronic F-68, whereas the use of formaldehyde alone resulted in significantly lower counts. Immunofluorescence labeling and analysis with the solid-phase cytometer of fixed natural samples from a bloom of P. parvum occurring in Lake Colorado in Texas gave cell counts that were close to those obtained by the traditional method of counting using light microscopy. These results show that a solid-phase cytometer can be used to rapidly enumerate natural P. parvum cells and that it could be used to detect other toxic algae, with an appropriate antibody or DNA probe.

Antibodies, Monoclonal↗

PubMed2005

Different marine heterotrophic nanoflagellates affect differentially the composition of enriched bacterial communities.

We studied the effects of predation on the cytometric and phylogenetic features of two enriched bacterial communities obtained from two cultures of marine heterotrophic nanoflagellates: Jakoba libera and a mixed culture of Cafeteria sp. and Monosiga sp. Protists were harvested by flow cytometric cell sorting and eight different treatments were prepared. Each bacterial community was incubated with and without protists, and we added two treatments with protists and the bacteria present after the sorting procedure (cosorted bacteria). The bacterial community derived from the culture of Jakoba libera had higher green fluorescence per cell (FL1) than that derived from the mixed culture of Cafeteria sp. and Monosiga sp. When the experiment began all treatments presented bacterial communities that increase in fluorescence per bacterium (FL1); after that the FL1 decreased when bacteria attained maximal concentrations; and, finally, there was a new increase in FL1 toward the end of the experiment. Cosorted bacteria of Jakoba libera had the same fluorescence as the bacterial community derived from this protist, while the bacteria derived from the mixed culture of Cafeteria sp. and Monosiga sp. was nearly twice as fluorescent than that of the parental community. All treatments presented a general decline of SSC along the incubation. Therefore, there was a small influence of protists on the cytometric signature of each bacterial community. However, each bacterial community preyed by Jakoba libera or the mixed culture of Cafeteria sp. and Monosiga sp. led to four different phylogenetic fingerprint. Besides, the final Communities were different from the fingerprint of controls without protists, and most of them diverge from the fingerprint of cosorted bacteria. Our results confirm that changes in the phylogenetic composition of marine bacterial communities may depend on the initial communities of both bacteria and protists.

Animals↗

PubMed2005

Beta-D-glucuronidase activity assay to assess viable Escherichia coli abundance in freshwaters.

AIMS: The relationships between the beta-D-glucuronidase (GLUase) activity, the abundance of culturable Escherichia coli and the number of viable E. coli were investigated in river and wastewater samples. METHODS AND RESULTS: GLUase activity was measured as the rate of hydrolysis of 4-methylumbelliferyl-beta-D-glucuronide. Culturable E. coli were enumerated by the most probale number (MPN) microplate method. Viable E. coli were estimated by fluorescent in situ hybridization (FISH) coupled with a procedure of viability testing (DVC-FISH procedure). Significant correlations were found between the log of GLUase activity and both, the log culturable E. coli and the log of viable E. coli. CONCLUSIONS: GLUase activity per viable E. coli gave a broadly constant value from low to highly contaminated waters while GLUase activity per culturable E. coli strongly increased at low contaminated waters because of an underestimation of the number of active E. coli by the culture-based method. SIGNIFICANCE AND IMPACT OF THE STUDY: GLUase activity is a reliable parameter for the rapid quantification of viable E. coli in waters.

Bacteriological Techniques↗

PubMed2003

A sensitive and rapid method to determine the viability of freeze-dried bacterial cells.

AIMS: To determine the potential use of flow cytometry for viability asssessment of freeze-dried bacterial cells. METHODS AND RESULTS: Escherichia coli CIP 54.8T and Vibrio metschnikovii CIP 104262 were analysed. The viability of freeze-dried cells resuspended in a nutrient broth was evaluated by culture whereas activity was determined by flow cytometry analysis of both esterase activity and cell death. Activity assessment by flow cytometry was found to be a rapid and good indicator of cell viability and was very efficient for quality control. For V. metschnikovii the fraction of active cells varies greatly depending on the freeze-drying procedure and within a given procedure. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial activity assessment by flow cytometry is very efficient for the control of freeze-dried cells.

Bacteriological Techniques↗

PubMed2002

Dynamic characteristics of Prochlorococcus and Synechococcus consumption by bacterivorous nanoflagellates.

We compared the characteristics of ingestion of Prochlorococcus and Synechococcus by the marine heterotrophic nanoflagellate Pseudobodo sp. and by a mixed nanoflagellate culture (around 3 microm in size) obtained from an open sea oligotrophic area. Maximum ingestion rate on Synechococcus (2.7 Syn flagellate(-1) h(-1)) was reached at concentrations of 5 x 10(5) Syn mL(-1) and decreased between 6 x 10(5) and 1.5 x 10(6) Syn mL(-1). In order to validate laboratory data, one set of data on Synechococcus grazing was obtained during a field study in the oligotrophic northeastern Mediterranean Sea. Ingestion rates by heterotrophic nanoflagellates were related to Synechococcus abundance in the water, and the feeding rate showed a clear diel rhythm with consumption being highest during the night, declining during the day hours, and being lowest at dusk. Ingestion rates on Prochlorococcus increased linearly for the whole range of prey density used (i.e., from 1 x 10(3) to 3 x 10(6) Proc mL(-1)), with maximum ingestion of 6.7 Proc flagellate(-1) h(-1). However, for prey concentrations in the range of 10(3)-10(5), which are usually encountered in aquatic systems, ingestion rates were significantly less than on Synechococcus. In our experiments, both Prochlorococcus and Synechococcus proved to be poor food items for support of nanoflagellate growth.

Animals↗

PubMed2001

A new approach to determine the genetic diversity of viable and active bacteria in aquatic ecosystems.

BACKGROUND: Discrimination among viable, active, and inactive cells in aquatic ecosystems is of great importance to understand which species participate in microbial processes. In this study, a new approach combining flow cytometry (FCM), cell sorting, and molecular analyses was developed to compare the diversity of viable cells determined by different methods with the diversity of total cells and active cells. METHODS: Total bacteria were determined by SYBR-II staining. Viable bacteria were determined in water samples from different sites by plate count techniques and by the direct viable count (DVC) method. Substrate-responsive cells (i.e., DVC(+) cells) were distinguished from nonresponsive cells (i.e., DVC(-) cells) by FCM and sorted. The genetic diversity of the sorted cell fraction was compared with the diversity of the total microbial community and with that of the culturable cell fraction by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments. The same approach was applied to a seawater sample enriched with nutrients. In this case, actively respiring cells (CTC+) were also enumerated by FCM, sorted, and analyzed by DGGE. RESULTS: The diversity of viable cells varied depending on the methods (traditional culture or DVC) used for viability assessment. Some phylotypes detected in the fraction of viable cells were not detectable at the community level (from total DNA). Similar results were found for actively respiring cells. Inversely, some phylotypes found at the community level were not found in viable and active cell-sorted fractions. It suggests that diversity determined at the community level includes nonactive and nonviable cells. CONCLUSION: This new approach allows investigation of the genetic diversity of viable and active cells in aquatic ecosystems. The diversity determined from sorted cells provides relevant ecological information and uncultured organisms can also be detected. New investigations in the field of microbial ecology such as the identification of species able to maintain cellular activity under environmental changes or in the presence of toxic compounds are now possible.

Bacteria↗

PubMed2001

Microbial community dynamics in Mediterranean nutrient-enriched seawater mesocosms: changes in the genetic diversity of bacterial populations.

A mesocosm experiment was performed to study the influence of nutrients on activity and diversity of bacterial assemblages from the Mediterranean Sea. Changes in the diversity of the predominant bacterial populations were monitored by DGGE fingerprinting of PCR products derived from 16S rRNA encoding genes. Fluctuations in the diversity of the most active populations was inferred by performing the DGGE fingerprinting on the basis of the cellular rRNA after reverse transcription and PCR amplification. DNA-derived DGGE patterns obtained from duplicate control and nutrient-enriched mesocosms showed differences in the development of the bacterial communities between control and nutrient-enriched experimental mesocosms. Multidimensional scaling analysis of the DNA-derived DGGE fingerprints indicated that duplicate treatments were reproducible. DNA- and RNA-derived DGGE fingerprints of bacterial assemblages changed over time, showing that the composition of the bacterial assemblages, as well as the most active bacterial populations changed during different phases of the incubation. Sequences of predominant DGGE bands in RNA-derived patterns were similar to 16S rRNA gene sequences of members of the alpha-, gamma- and delta-Proteobacteria and of the Cytophaga-Flavobacterium-Bacteroides phylum (CFB). Bands corresponding to Ruegeria-like bacteria and members of the CFB became especially dominant during the course of incubation, suggesting that these populations were important contributors to bacterial production and activity in the post-grazing phase of the experiment.

Journal Article↗

PubMed2001

Microbial community dynamics in Mediterranean nutrient-enriched seawater mesocosms: changes in abundances, activity and composition.

Quantitative and qualitative changes in bacterial communities from the Mediterranean Sea were compared in duplicate batch mesocosms with or without addition of inorganic nutrients. Methods including traditional microbial ecology techniques, molecular biology and flow cytometry were combined to determine abundances, production, cell size, activity, culturability and taxonomic diversity of bacterial cells. Addition of nutrients and confinement resulted in an increase of bacterial densities which were rapidly controlled by protozoan grazing. Changes in bacterial activity and morphology were observed during the growth phase of bacteria and under grazing pressure. The proportion of medium-size and culturable cells increased during the growth phase. These cells were preferentially consumed by grazers resulting in a strong limitation of bacterial production. As a consequence of the grazing pressure, large cells were produced and contributed to the remaining bacterial productivity after grazing. Grazing had an effect on the taxonomic composition of bacterial communities by preferentially eliminating gamma-Proteobacteria, alpha-Proteobacteria were preserved. It seems that some species from the genera Ruegeria and Cytophaga may have developed defence strategies to escape predation.

Journal Article↗

PubMed2001

Are the actively respiring cells (CTC+) those responsible for bacterial production in aquatic environments?

The 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) staining method is commonly and increasingly used to detect and to enumerate actively respiring cells (CTC+ cells) in aquatic systems. However, this method remains controversial since some authors promote this technique while others pointed out several drawbacks of the method. Using flow cytometry (FCM), we showed that CTC staining kinetics vary greatly from one sample to another. Therefore, there is no universal staining protocol that can be applied to aquatic bacterial communities. Furthermore, using (3)H-leucine incorporation, it was shown that the CTC dye has a rapid toxic effect on bacterial cells by inhibiting protein synthesis, a key physiological function. The coupling of radioactive labelling with cell sorting by FCM suggested that CTC+ cells contribute to less than 60% of the whole bacterial activity determined at the community level. From these results, it is clearly demonstrated that the CTC method is not valid to detect active bacteria, i.e. cells responsible for bacterial production.

Journal Article↗

PubMed2001

Does the high nucleic acid content of individual bacterial cells allow us to discriminate between active cells and inactive cells in aquatic systems?

The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems.

Bacteria↗

PubMed2000

Relationships among Bacterial Cell Size, Productivity, and Genetic Diversity in Aquatic Environments using Cell Sorting and Flow Cytometry.

The study of relationships between cell size and productivity is of key importance in microbial ecology to understand which members of natural aquatic communities are responsible for the overall activity and/or productivity. Flow sorting of microorganisms from different environmental samples was used to analyze the activity of bacterial cells depending on their biovolume. Bacterial cells from five different natural samples taken along the Mediterranean coast including fresh- and seawaters were incubated with tritiated leucine, then stained with SYTO 13 and sorted by flow cytometry according to their average side-angle-scattered (SSC) light. In all samples, a bell-shaped relationship was found between cell biovolume and activity, whereas activity of a given cell-size class varied between samples. In contrast, an inverse relationship was found between biovolumes and abundances. These results suggest that medium-sized cells with highest growth rates are probably submitted to intense grazing. For one sample, bacteria within five different size classes were sorted and the genetic diversity of cells within each sorted size class and that of the whole community were analyzed by the denaturing gradient gel electrophoresis (DGGE) method. The genetic diversity, as determined at the community level was highly represented into the pool of small cells, whereas only few species were present into larger cell subpopulations. The results suggest that only a few genotypes may be dominant within the largest and most productive cells. Furthermore, cell size polymorphism as well as heterogeneous cellular activities were found within some species.

Journal Article↗

PubMed2000

Use of fluorescent probes to assess physiological functions of bacteria at single-cell level.

A wide diversity of fluorescent probes is currently available to assess the physiological state of microorganisms. The recent development of techniques such as solid-phase cytometry, the increasing sensitivity of fluorescence tools and multiparametric approaches combining taxonomic and physiological probes have improved the effectiveness of direct methods in environmental and industrial microbiology.

Bacterial Physiological Phenomena↗

PubMed2000

Evaluation of ChemChrome V6 for bacterial viability assessment in waters.

The efficiency of ChemChrome B (CB) and ChemChrome V6 (CV6) dyes to stain viable bacterial cells in water was compared. Both dyes are fluorogenic esters converted to free fluorescein by esterase activity. The dyes were applied to a wide variety of bacterial species, including those poorly stained by CB, and to natural waters. Some species tested gave unacceptable low fluorescence intensities by being inefficiently or non-labelled with the CB. In contrast, CV6-stained bacteria were easily detected by both flow cytometry and solid-phase cytometry. As a consequence, higher viable cell counts were found with CV6 compared with CB in natural waters. Viable counts determined by CV6 staining were always higher than cfu counts. In contrast, respiring cell counts (CTC) were always lower than CV6 counts and, in the case of tap and mineral waters, they were lower than cfu counts.

Bacteria↗

PubMed2000

Current and future applications of flow cytometry in aquatic microbiology.

Flow cytometry has become a valuable tool in aquatic and environmental microbiology that combines direct and rapid assays to determine numbers, cell size distribution and additional biochemical and physiological characteristics of individual cells, revealing the heterogeneity present in a population or community. Flow cytometry exhibits three unique technical properties of high potential to study the microbiology of aquatic systems: (i) its tremendous velocity to obtain and process data; (ii) the sorting capacity of some cytometers, which allows the transfer of specific populations or even single cells to a determined location, thus allowing further physical, chemical, biological or molecular analysis; and (iii) high-speed multiparametric data acquisition and multivariate data analysis. Flow cytometry is now commonly used in aquatic microbiology, although the application of cell sorting to microbial ecology and quantification of heterotrophic nanoflagellates and viruses is still under development. The recent development of laser scanning cytometry also provides a new way to further analyse sorted cells or cells recovered on filter membranes or slides. The main infrastructure limitations of flow cytometry are: cost, need for skilled and well-trained operators, and adequate refrigeration systems for high-powered lasers and cell sorters. The selection and obtaining of the optimal fluorochromes, control microorganisms and validations for a specific application may sometimes be difficult to accomplish.

Animals↗

PubMed2000

Diversity of Salmonella strains isolated from the aquatic environment as determined by serotyping and amplification of the ribosomal DNA spacer regions.

Salmonella species are pathogenic bacteria often detected in sewage, freshwater, marine coastal water, and groundwater. Salmonella spp. can survive for long periods in natural waters, and the persistence of specific and epidemic strains is of great concern in public health. However, the diversity of species found in the natural environment remains unknown. The aim of this study was to investigate the diversity of Salmonella strains isolated from different natural aquatic systems within a Mediterranean coastal watershed (river, wastewater, and marine coastal areas). A total of 574 strains isolated from these natural environments were identified by both conventional serotyping and the ribosomal spacer-heteroduplex polymorphism (RS-HP) method (M. A. Jensen and N. Straus, PCR Methods Appl. 3:186-194, 1993). More than 40 different serotypes were found, and some serotypes probably mobilized from widespread animal-rearing activities were detected only during storm events. These serotypes may be good indicators of specific contamination sources. Furthermore, the RS-HP method based on the PCR amplification of the intergenic spacer region between the 16S and 23S rRNA genes can produce amplicon profiles allowing the discrimination of species at both serotype and intraserotype levels. This method represents a powerful tool that could be used for rapid typing of Salmonella isolates.

Bacterial Typing Techniques↗

PubMed1999

Effectiveness of CSE to counterstain particles and dead bacterial cells with permeabilised membranes: application to viability assessment in waters.

The CSE dye (Chemunex, Maisons-Alfort, France) was combined with an activity marker to improve bacterial activity assessment in natural waters. Its effectiveness to counterstain dead cells with permeabilised membranes was investigated on live and dead cells of a variety of strains from collections or isolated from the natural environment. Cells were killed by heat treatment. For all strains tested, the fluorescent dye showed an intense staining of killed cells having permeabilised membranes while no significant signal was detected when applied to live cells. Furthermore, the CSE dye had no toxicity on viable cells. Then, CSE was combined with the ChemChrome V6 dye (Chemunex) to assess the activity of bacterial cells in different waters. Both fluorescences were analysed simultaneously by solid-phase cytometry. The active cell counts were sometimes lower when both dyes were combined suggesting that CSE was able to counterstain cells having a residual esterase activity and compromised membranes. These cells were subtracted from the active cell counts determined with ChemChrome V6. In most samples, active cell counts were congruent with those determined by the direct viable count method.

Bacteria↗

PubMed1999

Coupling Bacterial Activity Measurements with Cell Sorting by Flow Cytometry.

> Abstract A new procedure to investigate the relationship between bacterial cell size and activity at the cellular level has been developed; it is based on the coupling of radioactive labeling of bacterial cells and cell sorting by flow cytometry after SYTO 13 staining. Before sorting, bacterial cells were incubated in the presence of tritiated leucine using a procedure similar to that used for measuring bacterial production by leucine incorporation and then stained with SYTO 13. Subpopulations of bacterial cells were sorted according to their average right-angle light scatter (RALS) and fluorescence. Average RALS was shown to be significantly related to the average biovolume. Experiments were performed on samples collected at different times in a Mediterranean seawater mesocosm enriched with nitrogen and phosphorus. At four sampling times, bacteria were sorted in two subpopulations (cells smaller and larger than 0.25 µm(3)). The results indicate that, at each sampling time, the growth rate of larger cells was higher than that of smaller cells. In order to confirm this tendency, cell sorting was performed on six subpopulations differing in average biovolume during the mesocosm follow-up. A clear increase of the bacterial growth rates was observed with increasing cell size for the conditions met in this enriched mesocosm.http://link.springer-ny.com/link/service/journals/00248/bibs/38n2p180.html

Journal Article↗