Patrick Monfort

Cell cycle characteristics and changes in membrane potential during growth of Escherichia coli as determined by a cyanine fluorescent dye and flow cytometry

Abstract A preliminary study on the optimization of the DiOC 6 (3) staining technique using flow cytometry for evaluating the membrane potential of Escherichia coli showed that DiOC 6 (3) was an adequate membrane potential probe. Flow cytometry was used to determine the growth curve by cell direct counts, to examine cell cycle characteristics by measuring DNA content using Hoechst 33342, and to observe changes in membrane potential using DiOC 6 (3) of E. coli in optimum culture conditions. The membrane potential increased abruptly from lag phase and the maximum value occurred before the mid-exponential phase at the end of 1 h 30 min of culture. At the same time, replication activity, as determined by measuring the number of DNA replication origins, reached its maximum. After the mid-exponential phase, while generation time was 23 min, membrane potential and DNA replication decreased steadily until the stationary phase, with cells using the replicated chromosome stocks. The rapid increase of membrane potential as early as the lag phase until just before the mid-exponential phase suggested that metabolic activity was maximum during this period to respond to energy supplies that were necessary, in part, for DNA replication.

Quantification of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae in French Mediterranean coastal lagoons.

Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae are human pathogens. Little is known about these Vibrio spp. in the coastal lagoons of France. The purpose of this study was to investigate their incidence in water, shellfish and sediment of three French Mediterranean coastal lagoons using the most probable number-polymerase chain reaction (MPN-PCR). In summer, the total number of V. parahaemolyticus in water, sediment, mussels and clams collected from the three lagoons varied from 1 to >1.1 × 10³ MPN/l, 0.09 to 1.1 × 10³ MPN/ml, 9 to 210 MPN/g and 1.5 to 2.1 MPN/g, respectively. In winter, all samples except mussels contained V. parahaemolyticus, but at very low concentrations. Pathogenic (tdh- or trh2-positive) V. parahaemolyticus were present in water, sediment and shellfish samples collected from these lagoons. The number of V. vulnificus in water, sediment and shellfish samples ranged from 1 to 1.1 × 10³ MPN/l, 0.07 to 110 MPN/ml and 0.04 to 15 MPN/g, respectively, during summer. V. vulnificus was not detected during winter. V. cholerae was rarely detected in water and sediment during summer. In summary, results of this study highlight the finding that the three human pathogenic Vibrio spp. are present in the lagoons and constitute a potential public health hazard.

Influence of temperature, salinity and E. coli tissue content on immune gene expression in mussel: results from a 2005-2008 survey.

Several bivalves, including mussels, suffered from mortalities particularly in summer. To look for the possible effect of environmental parameters on immune capacities, Mytilus galloprovincialis were collected monthly from August 2005 to July 2008 from the Palavas Laguna, French Mediterranean coast. Q-PCR was used to quantify the expression of three antimicrobial peptide genes (defensin, mytilin B and myticin B), in addition to lysozyme and HSP70. House keeping gene was 28S rRNA. Defensin, myticin B and lysozyme appeared more expressed in spring-summer than in winter. In contrast, HSP70 expression was higher in winter. Statistical studies using principal component analysis (PCA) and multiple regression models revealed positive influence of temperature on 28S rRNA, defensin, myticin B and lysozyme expressions, but not on mytilin B and HSP70. The positive influence was significant for defensin and lysozyme expression, but relationships cannot be quantified. Similarly, salinity appeared to influence defensin expression, but this relationship cannot be quantified neither. E. coli tissue content appeared without influence. Consequently, there was no clear relationship between environmental parameters and immune-related gene expressions, demonstrating anti-infectious capabilities cannot be evaluated using only the expression of such genes as markers.

Respective Roles of Culturable and Viable-but-Nonculturable Cells in the Heterogeneity of Salmonella enterica Serovar Typhimurium Invasiveness

ABSTRACT The existence of Salmonella enterica serovar Typhimurium viable-but-nonculturable (VBNC) cells is a public health concern since they could constitute unrecognized sources of infection if they retain their pathogenicity. To date, many studies have addressed the ability of S. Typhimurium VBNC cells to remain infectious, but their conclusions are conflicting. An assumption could explain these conflicting results. It has been proposed that infectivity could be retained only temporarily after entry into the VBNC state and that most VBNC cells generated under intense stress could exceed the stage where they are still infectious. Using a Radioselectan density gradient centrifugation technique makes it possible to increase the VBNC-cell/culturable-cell ratio without increasing the exposure to stress and, consequently, to work with a larger proportion of newly VBNC cells. Here, we observed that (i) in the stationary phase, the S. Typhimurium population comprised three distinct subpopulations at 10, 24, or 48 h of culture; (ii) the VBNC cells were detected at 24 and 48 h; (iii) measurement of invasion gene (hilA, invF, and orgA) expression demonstrated that cells are highly heterogeneous within a culturable population; and (iv) invasion assays of HeLa cells showed that culturable cells from the different subpopulations do not display the same invasiveness. The results also suggest that newly formed VBNC cells are either weakly able or not able to successfully initiate epithelial cell invasion. Finally, we propose that at entry into the stationary phase, invasiveness may be one way for populations of S. Typhimurium to escape stochastic alteration leading to cell death.

Haemolysin occurrence amongAeromonas hydrophila,Aeromonas caviae andAeromonas sobria strains isolated from different aquatic ecosystems

A total of 909 Aeromonas spp. isolates from different aquatic ecosystems were tested for haemolysin production by both sheep and horse blood agar-plate assays and by rabbit erythrocytes in broth assay. A comparison of these different methods was undertaken in order to appreciate their capacity to evaluate the haemolytic activity of Aeromonas spp. isolated from aquatic ecosystems. The haemolytic activity was associated particularly with A. hydrophila and A. sobria (about 95% of strains), whereas A. caviae did not produce haemolysin (about 95% of strains). A method suitable for use in routine diagnostic microbiology laboratories is proposed for quantifying both groups of A. hydrophila/A. sobria and A. caviae in environmental water.

Fatal disease mimicking leptospirosis in a dog, caused by Aeromonas hydrophila

A dog was treated for leptospirosis on clinical and epidemiological arguments. The amoxicillin treatment was not successful. Pure culture of Aeromonas hydrophila was then obtained from liver and kidney, indicating that the septicemia was due to this bacteria commonly found in waters.

Highly diverse recombining populations of Vibrio cholerae and Vibrio parahaemolyticus in French Mediterranean coastal lagoons.

Vibrio parahaemolyticus and Vibrio cholerae are ubiquitous to estuarine and marine environments. These two species found in Mediterranean coastal systems can induce infections in humans. Environmental isolates of V. cholerae (n = 109) and V. parahaemolyticus (n = 89) sampled at different dates, stations and water salinities were investigated for virulence genes and by a multilocus sequence-based analysis (MLSA). V. cholerae isolates were all ctxA negative and only one isolate of V. parahaemolyticus displayed trh2 gene. Most Sequence Types (ST) corresponded to unique ST isolated at one date or one station. Frequent recombination events were detected among different pathogenic species, V. parahaemolyticus, V. cholerae, Vibrio mimicus, and Vibrio metoecus. Recombination had a major impact on the diversification of lineages. The genetic diversity assessed by the number of ST/strain was higher in low salinity condition for V. parahaemolyticus and V. cholerae whereas the frequency of recombination events in V. cholerae was lower in low salinity condition. Mediterranean coastal lagoon systems housed V. cholerae and V. parahaemolyticus with genetic diversities equivalent to the worldwide diversity described so far. The presence of STs found in human infections as well as the frequency of recombination events in environmental vibrios populations could predict a potential epidemiological risk.

Rapid Proliferation of Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae during Freshwater Flash Floods in French Mediterranean Coastal Lagoons

ABSTRACT Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae of the non-O1/non-O139 serotype are present in coastal lagoons of southern France. In these Mediterranean regions, the rivers have long low-flow periods followed by short-duration or flash floods during and after heavy intense rainstorms, particularly at the end of the summer and in autumn. These floods bring large volumes of freshwater into the lagoons, reducing their salinity. Water temperatures recorded during sampling (15 to 24°C) were favorable for the presence and multiplication of vibrios. In autumn 2011, before heavy rainfalls and flash floods, salinities ranged from 31.4 to 36.1‰ and concentrations of V. parahaemolyticus, V. vulnificus, and V. cholerae varied from 0 to 1.5 × 103 most probable number (MPN)/liter, 0.7 to 2.1 × 103 MPN/liter, and 0 to 93 MPN/liter, respectively. Following heavy rainstorms that generated severe flash flooding and heavy discharge of freshwater, salinity decreased, reaching 2.2 to 16.4‰ within 15 days, depending on the site, with a concomitant increase in Vibrio concentration to ca. 104 MPN/liter. The highest concentrations were reached with salinities between 10 and 20‰ for V. parahaemolyticus, 10 and 15‰ for V. vulnificus, and 5 and 12‰ for V. cholerae. Thus, an abrupt decrease in salinity caused by heavy rainfall and major flooding favored growth of human-pathogenic Vibrio spp. and their proliferation in the Languedocian lagoons. Based on these results, it is recommended that temperature and salinity monitoring be done to predict the presence of these Vibrio spp. in shellfish-harvesting areas of the lagoons.

Characterization of the Dynamics of Microbiological and Chemical Contaminants in an Urban Catchment in South of France: From Field Data Collection to Modelling

Densely urbanized areas generate a wide range of contaminants exerting pressures on the quality of downstream rivers. As urban areas are in permanent extension, assessing the potential evolution of water quality and the effects of mitigation measures is a real challenge. Modelling tools could be helpful in decision making but require sound field data for calibration steps. The present study focuses on the urban river Lez that crosses the area of Montpellier city. Due to the Mediterranean climate, extreme precipitations intensify the transfer of contaminants by generating land runoff and significant sewer overflow. The spatiotemporal variability in microbiological and chemical contamination of the Lez river and its tributaries is characterized through field campaigns. Three low flow campaigns and a flood event are presented here, with a focus on the most urbanized sub-catchment: Verdanson. Hydrological conditions are shown to have a strong impact on the water quality in terms of concentrations of fecal indicator bacteria, organotins, trace metals and polycyclic aromatic hydrocarbons. As a first step towards the modelling of the water quality during a flood event, a sensitivity analysis was performed to test how land cover could be properly be taken into account in the modelling process. The analysis reveals that discretizing the catchment into land cover-homogeneous subcatchments enhances the suitability of water quality model outputs, keeping good outputs of the hydrological model. Consequently, a rigorous description of land cover is required to properly assess the impacts of territorial dynamics on water quality.