Development of a DGGE method to explore Legionella communities.

Legionella risk assessment is nowadays based on the presence and concentration of either Legionella pneumophila or Legionella spp. Many species of Legionella can cause Legionnaires' disease, indeed about half of the known species have been associated with infection. The aim of this work was to develop a method to assess the composition of the Legionella species community in an environmental sample in order to have a better understanding of the contamination of the ecosystem by pathogenic strains. The method is based on the comparison of PCR-DGGE profile of DNA sample with a database consisting in DGGE profiles of Legionella species. Such a database includes all pathogenic Legionella strains. In order to homogenize and normalize the different DGGE fingerprint, a reference marker has been built and added during DGGE gel analysis. This study gives a valuable advance in the methods available for the understanding of Legionella contamination of water environments.

Assessment of poly-L-lysine dendrigrafts for virus concentration in water: use of MS2 bacteriophage as proof of concept.

AIMS: Virus detection has often been difficult due to a low concentration in water. In this study, we developed a new procedure based on concentration of virus particles on an innovative support: poly-L-lysine dendrigrafts (DGL), coupled with directed nucleic acid extraction and real-time PCR quantification. METHODS AND RESULTS: This method was evaluated using the bacteriophage MS2 as a model virus. This virus exhibited the size and structural properties of human pathogenic enteric viruses and has often been used to assess new supports of concentration. Moreover, this bacteriophage is also a faecal contamination indicator. In this study, many water filtration conditions were tested (volume of water, concentration, etc.), and more than 80% of bacteriophage were recovered after filtration on polymer, in most conditions. We demonstrated that the method was linear (slope = 0·99 ± 0·04 and Y intercept when x = -0·02 ± 0·28), valid (as manipulators, tested concentrations, volumes of sample and batch of polymer did not have any influence on concentration) and sensitive (allowing to concentrate up to 16,600-fold 1 l of sample and to detect and quantify down to 750 GC l(-1) and 7500 GC l(-1), respectively). CONCLUSIONS: To conclude, this support exhibits high interest to retain viruses and to allow to detect low concentration of virus in water. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives valuable advance in the methods of concentration and diagnosis of virus in water.

The characterization and certification of a quantitative reference material for Legionella detection and quantification by qPCR.

AIMS: The characterization and certification of a Legionella DNA quantitative reference material as a primary measurement standard for Legionella qPCR. METHODS AND RESULTS: Twelve laboratories participated in a collaborative certification campaign. A candidate reference DNA material was analysed through PCR-based limiting dilution assays (LDAs). The validated data were used to statistically assign both a reference value and an associated uncertainty to the reference material. CONCLUSIONS: This LDA method allowed for the direct quantification of the amount of Legionella DNA per tube in genomic units (GU) and the determination of the associated uncertainties. This method could be used for the certification of all types of microbiological standards for qPCR. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of this primary standard will improve the accuracy of Legionella qPCR measurements and the overall consistency of these measurements among different laboratories. The extensive use of this certified reference material (CRM) has been integrated in the French standard NF T90-471 (April 2010) and in the ISO Technical Specification 12 869 (Anon 2012 International Standardisation Organisation) for validating qPCR methods and ensuring the reliability of these methods.

Water reuse for urban landscape irrigation: aspersion and health related regulations.

The Mediterranean seaside resort of Le Grau du Roi includes 40 hectares of landscaped areas spray irrigated with river water supplied through a separate network. Wastewater collected from several municipalities is treated in an activated sludge wastewater treatment plant (WWTP) and polished in waste stabilization ponds (WSPs). Planned substitution of treated wastewater for river water is hindered by spray irrigation prohibition within a 100 m distance from houses and recreational areas. WWTP and WSP effluents were monitored for pathogens with a particular attention to Legionella in Spring and Summer 2006. Helminth eggs, salmonellae and enteroviruses were never detected neither in WWTP effluent nor in the ponds. Legionella spp content was slightly higher or of the order of magnitude of river water contents. Regarding Legionella pneumophila contents, WSP effluent did not significantly differ from the river water. E.coli and enterococci contents in WSP effluents complied with the "excellent quality" criteria of the European Directive for coastal bathing waters. Therefore, substituting WSP effluents to river water is unlikely to alter health risks related to spray irrigation and, in this case, the buffer zone required by the French water reuse guidelines appears being short of support.

The microbial signature of drinking waters: myth or reality?

This paper presents a new software developed for analyzing single strand conformation polymorphism (SSCP) electrophoresis patterns delivered by the genetic analyzer ABI310 (Applied Biosystems). SSCP is a molecular typing technique based on the PCR amplification of microbial 16S rDNA and used for the monitoring of complex microbial ecosystems dynamics. The software--a home-made MATLAB toolbox called MODIMECO--developed for the analysis of SSCP patterns is presented. MODIMECO includes a number of basic signal processing abilities as well as largely used statistical tools such as the well known principal component analysis. The use of the SSCP for assessing the hypothesis of the existence of a microbial signature of drinking waters illustrates the typical advantages of using such software tools. Results are discussed and conclusions drawn.

Comparison of methods for measurement of organic compounds at ultra-trace level: analytical criteria and application to analysis of amino acids in extraterrestrial samples.

The need for criteria to compare different analytical methods for measuring extraterrestrial organic matter at ultra-trace levels in relatively small and unique samples (e.g., fragments of meteorites, micrometeorites, planetary samples) is discussed. We emphasize the need to standardize the description of future analyses, and take the first step toward a proposed international laboratory network for performance testing.

Time and dose dependent effect of indomethacin on BCG induced PMN migration in mice.

The effect of three doses of indomethacin (Indo) on BCG-induced PMN migration at different times of day was studied in Swiss mice kept on a lighting regimen of LD 12:12, with L from 07:00 to 19:00. Experimental granulomas were induced by subcutaneous implantation of BCG-impregnated cell traps for a time span of 480 min. Doses of 1, 3 and 9 mg/kg of Indo were given orally one hour before trap implantation, at 01:00, 05:00, 09:00, 13:00, 17:00 and 21:00 hr. In sham animals, the maximal PMN count occurred at 17:00 hr. In treated mice, Indo increased or decreased the number of PMN/mm2 as a function of time of administration. Cell migration was inhibited at 17:00 hr by all 3 Indo doses, while the number of PMN in the cell trap increased at 21:00 hr. Various dose-effects were obtained at the other times of day. Several hypotheses are proposed to explain the conflicting data. The results indicate the importance of the time of drug administration in biology.

Effects of castration, Depo-testosterone and cyproterone acetate on lymphocyte T subsets in mouse thymus and spleen.

The effects of testosterone on the relative proportion of Thy 1.2, CD4 (L3T4) and CD8 (Lyt-2) cells in thymus and spleen were studied after castration and administration of Depo-testosterone (DT) separately or together with cyproterone acetate (CA) (an antiandrogen) in BDF1 mice. Injection of 0.5 mg/100 g body weight of DT during 2 weeks decreased significantly the number and proportion of double positive (DP) (CD4+ CD8+) and increased the percentage of single positive (SP) CD4+ (CD4+ CD8-), whereas there was a slight decrease in the Thy 1.2+ cells in the thymus. In parallel, we observed an increase in CD8+ (CD4- CD8+) cells in the spleen. The androgen deprivation after 3 weeks of castration induced a decrease in the percentage of CD4+ cells in thymus and both CD4+ and CD8+ cells in spleen. Injection of CA (0.5 mg/100 g body weight) had the same qualitative effects as DT on the proportion of lymphocyte T subsets in castrated mice. However, the combined activities of DT and CA were greater than either alone. These data indicate the main role of testosterone in the distribution of CD4+ and CD8+ cells in male mice. The similar effects of CA and DT in the lymphoid organs may suggest a difference between androgen receptors of sexual and lymphoid organs.

Temporal study of carrageenan-induced PMN migration in mice.

The time course of polymorphonuclear neutrophil (PMN) migration in an experimental inflammatory granuloma induced by carrageenan in the mice was studied at 6 different times of the day. Compared to saline, the number of PMN increased as soon as 240 min and reached high level at 480 min when migration was induced by carrageenan. The data showed circadian variations in the time course of cell migration. After 240 and 360 minutes of migration, the number of PMN was highest in the cell trap implanted at 09.00 whereas minimum values were obtained at 01.00. After 480 minutes of implantation, the PMN migration was maximal when the carrageenan experiment was performed at 05.00 and minimum values were obtained at 13.00. Thus, a significant rhythm was demonstrated in the time course of PMN migration.

Nycthemeral variations on LPS- and BCG-induced PMN migration in normal mice.

This study was performed to determine whether temporal variations could exist in polymorphonuclear neutrophil (PMN) migration induced by LPS or BCG in mice. LPS- and BCG-impregnated cell traps were implanted at 6 different times of the day and removed after 480 min. The PMN number per square unit (mm2) was counted and the results were expressed as means +/- S.E. in 8-10 mice. The results showed that nycthemeral variations occurred in both LPS- and BCG-induced PMN migration. Between 5.00 and 9.00 h a maximum value of 4210 +/- 270 and 1920 +/- 486 PMN/mm2 were obtained with BCG and LPS respectively; at 17.00 h a minimum value of 1300 +/- 270 with BCG and 396 +/- 127 PMN/mm2 with LPS were observed. There was no significant nycthemeral variation in saline-induced PMN migration.

Influence of BCG administration time on the in-vivo migration of leukocytes.

The temporal variation in the migration of polymorphonuclear leukocytes (PMN) induced by live BCG was studied in the mouse. Ten microliter of a 5 X 10(6) live BCG/ml suspension or sterile saline were placed on a cell trap immediately before its subcutaneous implantation at different clock times: 0100, 0500, 0900, 1300, 1700 and 2100 in animals synchronized with L(0700-1900): D(1900-0700). Eight hours later, the cell trap was removed, prepared for histological identification and counted. PMN counts in the cell trap were maximal 480 min after implantation. Non-specific migration was thought to occur and the peak value of leukocytes of 22.8 +/- 6.1 cells/10,000 micron 2 was obtained when the saline cell-trap was implanted at 0500. In the BCG-treated mice, a circadian rhythm was observed in the migration of leukocytes. The acrophase was at 1700. The results support the hypothesis that the circadian stage of antigen encounter influences the magnitude of the immune response.

Monthly changes in the effect of BCG on the migration of polymorphonuclear leucocytes.

Monthly changes in the migration of polymorphonuclear leucocytes (PMN) produced by subcutaneous implantation of BCG-impregnated rayone-made disks was examined over a period of 15 months. In experiments carried out at 90h00, the highest PMN count of 40.2 +/- 8.4 PMN/10,000 micrometers2 10,000 micrometers2 was obtained in April. Mechanisms that could explain the circannual variation are suggested.