Molecular typing of the Legionella pneumophila population isolated from several locations in a contaminated water network.

The RAPD (random amplified polymorphic DNA) technique has been developed for the molecular typing of Legionella in order to characterise the populations of hot water systems. During this study, 22 primers were tested and the four most informative ones were selected. The optimisation of the PCR conditions allowed the setting up of a powerful discriminative genotyping method. Moreover, the definition of a quality management method allowed definition of the key steps and the number of replicates to ensure reproducibility of the RAPD pattern. The RAPD was used to study the hot water network of a building. Legionella colonies (91) were isolated from seven locations and genotyped. The diversity of the population in one sample could vary from one to seven different strains. The study of the traceability showed that, in most of the cases, different populations could be present at different locations of the same network.

Optimization of stir bar sorptive extraction applied to the determination of odorous compounds in drinking water.

The off-flavour compounds 2-methylisoborneol (MIB), geosmin, 2,4,6-trichloroanisole, 2,3,6-trichloroanisole, 2,3,4-trichloroanisole and 2,4,6-tribromoanisole were analyzed in water samples by Stir Bar Sorptive Extraction (SBSE) followed by on-line thermal desorption (TD)-capillary GC/MS. Quantification was performed using MS in the single ion monitoring mode (SIM) with 2,4,6-trichloroanisol-D5 as internal standard. Quantification limits are 0.1 ng/l to 0.2 ng/l for the haloanisoles, 0.5 ng/l for geosmin and 1 ng/l for MIB. The relative standard deviations at the quantification limit are ranging from 7 to 14.6%. SBSE-recovery was evaluated by spiking real water samples and varied from 87 to 117%. More than twenty samples per day can be analyzed by SBSE-TD-capillary GC-MS. The same technique in combination with olfactometry was used to elucidate unknown odorous compounds in water samples.

Rapid and automated detection of fluorescent total bacteria in water samples.

Traditional methods for the detection and enumeration of bacteria in water samples are growth-based and require several days to obtain the result. New techniques which reduce the time of analysis have been developed. The objective of this work was to test a rapid method for the detection and enumeration of total viable bacteria using direct fluorescent labelling and detection by laser scanning. This method (referred to as TVC for Total Viable Count) was compared to the R2A culture method and the cyano-ditolyl-tetrazolium chloride (CTC) staining method for the analysis of samples before the final chlorination (after GAC filtration) and drinking water samples. For the comparison of TVC and CTC, the outcome depends on the water type: for samples after GAC filtration, TVC counts were significantly lower than CTC counts by up to 2 log10 orders of magnitude. For chlorinated water samples, TVC counts were not significantly different from CTC counts. The comparison of TVC and R2A showed that TVC counts could be lower than R2A counts or equivalent depending on the type of water. For drinking water, the TVC method proved to yield results equivalent to those of the R2A method. The TVC method requires much shorter time frame than others. It is also simple to use and allows the analysis of large volumes (100 ml) of drinking water.

Analysis of off-flavors in the aquatic environment by stir bar sorptive extraction-thermal desorption-capillary GC/MS/olfactometry.

The off-flavor compounds 2-methylisoborneol (MIB), geosmin, 2,4,6-trichloroanisole, 2,3,6-trichloroanisole, 2,3,4-trichloroanisole, and 2,4,6-tribromoanisole were analyzed in water samples by stir bar sorptive extraction (SBSE) followed by on-line thermal desorption (TD) capillary GC/MS. Quantification was performed using the MS in the single-ion-monitoring mode (SIM) with 2,4,6-trichloroanisole-D(5 )as internal standard. Quantification limits are 0.1-0.2 ng L(-1) for the haloanisoles, 0.5 ng L(-1) for geosmin, and 1 ng L(-1) for MIB. The relative standard deviations at the quantification limit ranged from 7 to 14.6%. SBSE recovery was evaluated by spiking real water samples and varied from 87 to 117%. More than twenty samples per day can be analyzed by SBSE-TD-capillary GC-MS. The same technique in combination with olfactometry was used to elucidate unknown odorous compounds in water samples.

Laser scanning detection of FISH-labelled Escherichia coli from water samples.

The development of rapid and accurate methods for the detection and quantification of bacteria without cultivation is of increasing importance for water monitoring. The aim of this study was to develop a solid phase cytometry detection method for DVC-FISH labelled Escherichia coli cells. In order to allow Stomatic detection with ChemScan RDI, the fluorescein-tyramide was combined with an oligonucleotide probe directly labelled with horseradish peroxidase to increase the fluorescence intensity. The method developed was tested for the enumeration of pure cultures, for GAC-filtered and drinking water samples. The method, which appeared to be equivalent to the culture method, was less sensitive than the DVC-FISH method followed by microscopic analysis. Research is underway to further optimise the labelling conditions.

Application of laser scanning cytometry followed by epifluorescent and differential interference contrast microscopy for the detection and enumeration of Cryptosporidium and Giardia in raw and potable waters.

AIMS: The main goal of this study was to validate a new laser scanning cytometry method (ChemScanRDI) that couples immunofluorescence detection with differential interference contrast (DIC) confirmation, against manual microscopic enumeration of Giardia and Cryptosporidium (oo)cysts. This study also assessed the basic performance of the new Association Française de Normalisation (AFNOR) NF T 90-455 method for Giardia and Cryptosporidium (oo)cyst enumeration with respect to (oo)cyst yield, linearity, repeatability, influence of turbidity and detection limit in raw and potable waters. METHODS AND RESULTS: The new standard method relies on cartridge (Envirocheck) filtration, immunomagnetic separation purification, immunofluorescence staining and detection followed by DIC confirmation. The recovery was 30-50% for both parasites at seeding levels from 30 to 230 (oo)cysts. The method is linear from 0 to around 400 seeded (oo)cysts and the yield does not significantly vary for turbidity levels from 10 to 40 Formazin Nephelometric Units (FNU). The results were obtained using manual microscopic enumeration of the (oo)cysts. The ChemScanRDI yielded counts that were at least equivalent to those obtained using manual microscopy for both parasites in raw and potable water concentrates, for seeding levels of 10-300 or 10-100, respectively. The purification and labelling method proposed by the supplier of theChemScanRDI (Chemunex) reached very similar recoveries to the AFNOR protocol (70-86% in both cases). CONCLUSIONS: Laser scanning cytometry can be used as a more standardized alternative to manual enumeration as part of the new AFNOR standard method. SIGNIFICANCE AND IMPACT OF THE STUDY: By using laser scanning cytometry instead of manual microscopy, laboratories could circumvent the limitations of manual microscopy, namely: low sample throughput, operator subjectivity and operator fatigue. The study further supports the drive to incorporate laser scanning cytometry in the standard methods for Giardia and Cryptosporidium enumeration.

Bacterial removal from inexpensive portable water treatment systems for travelers.

BACKGROUND: There are many personal portable water treatment systems for travelers on the market, including chemical agents, iodine resin purifiers and filters. However, information on the real efficacy of these systems in the field is often lacking. We have therefore estimated the capabilities of several inexpensive personal portable water treatment systems for travelers to remove bacteria in various situations of water quality, using stressed indigenous strains of bacteria. METHODS: Four chemical agents (Drinkwell chlorine, Hydroclonazone, Aquatabs, 2% iodine in ethanol), two iodine resin purifiers (the straw PentaPure Outdoor M1-E, the PentaPure Traveler purifying and filtration system) and four filters (the flexible bottle Pres2Pure, the hand-pump filters Mini Ceramic, First Need Deluxe and WalkAbout) were evaluated in triplicate using both turbid and clear water at 25 degrees C. Bacteria were counted by conventional culturing techniques, colorimetric and fluorescent assays of coliforms and Escherichia coli enzyme activities (Colilert)/Quantitray method), and viable but not culturable bacteria were assessed quantitatively by 5-cyano-2,3-dilotyl-tetrazolium staining. RESULTS: The best systems were the three hand-pump filters, Mini Ceramic, First Need Deluxe, and WalkAbout. All had a submicron filtration element that completely removed 3 log (99.9%) or more of viable bacteria, and no coliforms or E. coli were detected in the effluent. The PentaPure Traveler removed more than 99.3% of the viable bacteria. The only chemical agents that gave a bacterial inactivation of over 2 log in clear water were the Drinkwell chlorine, the Aquatabs, and the 2% iodine in ethanol. The three other devices, Hydroclonazone, Outdoor M1-E, and Pres2Pure, performed poorly, as coliforms and E. coli were detected in the treated water by the Colilert method. The chemical agents and the iodine resin straw performed poorly on raw river water; coliforms and E. coli were detected in the treated water. CONCLUSIONS: These data demonstrate the differences between the systems tested. The effectiveness of other devices on the market should also be tested, so as to help travelers and hikers select the most appropriate portable water treatment system.

Octahedral deformations and cationic displacements in the ferroelectric PbHf(0.8)Ti(0.2)O(3): a neutron powder diffraction study from 10 to 770 K

Neutron powder diffraction data, collected over the temperature range 10-770 K, have been analysed in order to make a detailed characterization of the sequence of phase transitions occurring in the Hf-rich ferroelectric PbHf(0.8)Ti(0.2)O3, titanium hafnium lead oxide. Over the whole temperature range this compound undergoes two phase transitions, which involve cationic displacements and octahedral deformations (tilt and/or distortion) leading to strongly distorted perovskite-type structures. The first transition appears around 415 K between two ferroelectric rhombohedral phases: a low-temperature nonzero-tilt phase F(RL) (space group R3c) and an intermediate zero-tilt phase FRH (space group R3m). The second one, detected around 520 K, is associated with a ferroelectric to-paraelectric transition between the FRH phase and the Pc cubic phase (space group Pm3m). From high-resolution neutron powder diffraction data (diffractometer 3T2-LLB, Saclay, France, lambda = 1.2251 A), the crystallographic structure of the three successive phases has been accurately determined at the following temperatures: T = 10 K (FRL): space group R3c, Z = 6, a(hex) = 5.7827 (1), c(hex) = 14.2702 (4) A, V(hex) = 413.26 (2) A3; T = 150 K (F(RL)): space group R3c, Z = 6, a(hex) = 5.7871 (1), C(hex) = 14.2735 (4) A, V(hex) = 413.98 (3) A3; T = 290 K (FRL): space group R3c, Z = 6, a(hex) = 5.7943 (1), C(hex) = 14.2742 (5) A, V(hex) = 415.04 (3) A3; T = 440 K (F(RH)): space group R3c, Z = 6, a(hex) = 5.8025 (1), c(hex) = 14.2648 (4) A, V(hex) = 415.94 (3) A3; T = 520 K (Pc): space group Pm3m, Z = 1, a(cub) = 4.1072 (2) A, V(cub) = 69.29 (1) A3. In addition, a neutron powder thermodiffractometry experiment, performed between 290 and 770 K (diffractometer D1B-ILL, Grenoble, France, lambda = 2.533 A), has been used to study in situ the temperature-induced phase transitions. From sequential Rietveld refinements, the temperature dependence of the cation displacements and the rotation and/or distortion of oxygen octahedra was derived.

Temperature-dependent neutron powder diffraction evidence for splitting of the cationic sites in ferroelectric PbHf0.4Ti0.6O3.

Temperature-dependent neutron powder diffraction experiments (diffractometer 3T2-LLB, Saclay, France, lambda = 1.227 Å) have been performed on the perovskite-like lead hafnate titanate PbHf(0.4)Ti(0.6)O(3). This compound belongs to the solid solution denoted PHT, which derives from the well known ferroelectric PZT series. It exhibits a ferroelectric-to-paraelectric phase transition around 620 K, between the low-temperature tetragonal phase and the high-temperature cubic phase. The tetragonal structure of the ferroelectric phase has been refined at 10 and 300 K using a Rietveld-type method: space group P4mm with Z = 1; a(t) = 3.999 (1), c(t) = 4.120 (1) Å, c/a = 1.030, V = 65.89 Å(3) at 10 K; a(t) = 4.012 (1) and c(t) = 4.100 (1) Å, c/a = 1.022, V = 65.99 Å(3) at 300 K. The cubic structure of the paraelectric phase has also been refined at 720 K: space group Pm3;m, Z = 1, a(c) = 4.046 (1) Å, V = 66.23 Å(3). Cation displacements and oxygen-octahedra elongations have been observed as a function of temperature. Evidence for peculiar behaviour associated with the relative shifts of the Hf and Ti atoms (thought until now to be on the same crystallographic site) was found through an anomaly of the mean-square atomic displacements of the Hf/Ti pseudo-nucleus. The PDF Nos for PbHf(0.4)Ti(0.6)O(3) are 48-49-9 and 48-49-10.

Influence of pH, nitrogen and phosphorus sources on the production of hepatitis B virus pre-S2 antigen by Hansenula polymorpha.

Experimental design techniques were used to study the influence of the composition of the culture medium on the production of hepatitis B virus pre-S2 antigen by the methylotrophic yeast Hansenula polymorpha. pH, phosphoric acid, ammonium chloride and yeast extract concentrations were selected as experimental factors and their influence was investigated using Central Composite design techniques. The results indicated that antigen yield was maximized at high pH and in a culture medium containing both ammonium chloride and yeast extract. Phosphoric acid was found to have a detrimental effect on antigen production. This study allowed a 50% increase in antigen production in a medium in which the yeast extract concentration was decreased to 32 g/l. These optimal conditions have been confirmed with an octagonal design experiment. Moreover, it was shown that the antigen produced was very stable up to at least 8 days after induction and that the yeast extract concentration could be lowered to 22 g/l without appreciable effect on antigen yield. The increase in antigen production was not due to an increase in cell biomass, since no correlation could be found between these two parameters. The newly defined culture medium should allow a greatly increased antigen production at the fermentor level, at a lower cost and with minimal operational problems.

Peptidoglycan biosynthesis in Escherichia coli: variations in the metabolism of alanine and D-alanyl-D-alanine.

The in vivo functioning of the alanine/D-alanyl-D-alanine pathway of Escherichia coli was investigated by determining precursor pool levels and specific enzyme activities under various growth conditions. Cells grown on D- or L-alanine showed several remarkable features compared with cells grown on other carbon sources: 10-fold higher values of the D-alanyl-D-alanine and the UDP-MurNAc-pentapeptide pools, a 240-fold increase of the alanine racemase activity, and the absence of bacteriolysis after treatment with D-cycloserine at high concentrations (50 micrograms ml-1). In cells grown on glucose, D-cycloserine (1 micrograms ml-1) led to depletion of the D-alanyl-D-alanine pool and to lysis, which was efficiently antagonized by chloramphenicol. A threefold increase of the dipeptide pool was observed when cells were treated with chloramphenicol alone. The alanine racemase activity was lowest in glucose-grown cells and the D-alanine:D-alanine ligase and D-alanyl-D-alanine-adding activities were the same whatever the carbon source. Molecular masses of 53-56 kDa and 56-60 kDa were estimated for the partially purified inducible alanine racemase and D-alanine:D-alanine ligase respectively.

Some factors influencing the proportion of periplasmic hepatitis B virus pre-S2 antigen in the recombinant yeast Hansenula polymorpha.

A central composite design (CCD) was used to evaluate, for the purpose of future process optimization, the influence of pH, yeast extract and ammonium chloride concentrations on the proportion of periplasmic hepatitis B pre-S2 antigen in the recombinant yeast Hansenula polymorpha. Each factor was tested at five levels, and a second order polynomial model for the proportion of periplasmic antigen was fitted to the treatment combinations. pH showed the greatest effect: the proportion of periplasmic antigen was greatly increased at the higher pH levels. At the higher pH levels used, the proportion of periplasmic antigen was enhanced by a high concentration of ammonium chloride. Additional experiments have confirmed both the validity of the selected model and the optimal conditions found. A significant correlation was found between the proportion of periplasmic antigen and the total yield of antigen. These results indicated that it should be possible to modulate the distribution of the pre-S2 antigen between the periplasm and the cytoplasm of the yeast.

Fermentation study for the production of hepatitis B virus pre-S2 antigen by the methylotrophic yeast Hansenula polymorpha.

Various physico-chemical parameters have been studied in order to improve the production of hepatitis B virus pre-S2 antigen (middle surface antigen) by the methylotrophic yeast Hansenula polymorpha. Antigen production was done in two steps: first, production of cells on glycerol (Phase 1), followed by induction of antigen expression with methanol (Phase 2). Dense cultures of H. polymorpha, equivalent to 35-40 g/l (dry weight), were readily obtained in small fermenters using minimal medium containing glycerol as carbon source. Antigen expression in this minimal medium, after induction with methanol, was however, low and never exceeded 1.6 mg/l of culture. Antigen production was greatly enhanced by adding complex organic nitrogen sources along with methanol at induction time; yeast extract was the best of all the sources tested. In shake flasks, antigen production was proportional to yeast extract concentration up to 7% (w/v) yeast extract, it became clear the the nutritional conditions for good antigen expression were different from those for good biomass production. The effects of yeast extract were reproduced in small fermenters: antigen levels reached 8-9 mg/l in medium containing 6% (w/v) yeast extract during induction with methanol. The mechanisms of yeast extract's effects are still unknown but are probably nutritional. The recombinant H. polymorpha strain produced both periplasmic and intracellular antigen. The periplasmic antigen was shown to be present as 20-22-nm particles and was therefore immunogenic. Immunoblotting indicated that part of the pre-S2 antigen was present as a 24-kDa degradation product. These studies have led to a 140-fold increase in volumetric productivity of antigen and to a 4.6-fold increase in specific production.

Embryonic testicular regression syndrome and severe mental retardation in sibs.

The embryonic testicular regression syndrome associated with severe mental retardation is reported in three 46,XY sibs each of whom has a 46,XY chromosome complement. A fourth sib, a sister, also is severely retarded mentally; her chromosome complement is 46,XX. The 46,XY individuals, who were raised as females, presented varying degrees of genital ambiguity, indicating that their gonadal activities had been arrested at different times during embryogenesis. No trace of gonadal tissue could be found in either patient. The coincidence of the embryonic testicular regression syndrome and severe mental retardation in the same sibship is discussed.

[Trisomy 21 by mirror duplication 46,XX,psu dic(21)ter rea (21q21q) (author's transl)]. / Trisomie 21 par duplication en miroir 46,XX,psu dic(21)ter rea(21q21q).

"Mirror image" duplication of chromosome 21 -- 46,XX,pter dic(21)ter rea(21q21q) -- was observed in a patient with the complete phenotype of trisomy 21 and a ses-sesquialtère de la SOD1.

[Dicentric Y chromosome in a male pseudohermaphrodite 45,X/46,X, dic (Y)/47, XYY]. / Chromosome Y dicentrique chez un pseudohermaphrodite masculin, [45,X/46,X, dic (Y)/47, XYY].

The mosaicism 45,X/46,XY,terrea(Y,Y)(pterpter)/47,XYY was observed in an 8-month-old child with male pseudohermaphroditism. The presence of a 47,XYY population points to a post-zygotic origin of the rearrangement. The loss of Yp material is in favor of localization of masculinization factor(s) to the proximal segment of Yq. Twenty-two relevant observations reported in the literature previously are discussed.

[Distal trisomy 15q]. / Trisomie 15q distale.

A patient with distal 15q trisomy resulting from malsegregation of a maternal t(13;15)(q33;q21.2) showed the following symptoms: micro-dolichocephaly, palpebral fissures slightly oriented downwards and outwards, a large nose, pronounced micrognathia, prominent authelices, ligamental abnormalities, osseous malformations evocative of diastrophic dwarfism, severe congenital heart defect, and profound encephalopathy. He died at five months of age. This observation is compared with two others from the literature.