Raw and processed data used in the simultaneous analysis of electrical characteristics and microstructure of crystallised PEDOT:PSS based OECTs under strain.

Here is presented raw and analysed data collected during study of the evolution, with uniaxial stretching, of the electrical and microcrystalline characteristics of polystyrene sulfonate doped poly(3,4-ethylenedioxythiophene) (PEDOT:PSS) organic electrochemical transistors (OECTs). X-ray diffraction data from GIWAXS measurements of the PEDOT:PSS material, performed at the SOLEIL light source are presented in raw and partially analysed forms. Current-voltage data, collected concurrently with the GIWAXS data, are also presented, and the evolution of the transconductance of the OECT devices with stretching is shown. GIWAXS data are only examined along the qz specular reflection ridge, and scans along this ridge are extracted and presented. However, the off-specular data may also be of interest to readers and is therefore made available here in its entirety.

Monitoring of cell layer coverage and differentiation with the organic electrochemical transistor.

Electrical, label-free monitoring of cells is a non-invasive method for dynamically assessing the integrity of cells for diagnostic purposes. The organic electrochemical transistor (OECT) is a device that has been demonstrated to be advantageous for interfacing with biological systems and had previously been shown to be capable of monitoring electrically tight, resistant, barrier type tissue. Herein, the OECT is demonstrated not only for monitoring of barrier tissue cells such as MDCK I, but also for other, non-barrier tissue adherent cells including HeLa cells and HEK epithelial cells. Transistor performance, expressed as transconductance (gm) is measured as a function of frequency; barrier tissue type cells are shown to have a more abrupt drop in transconductance compared to non-barrier tissue cells, however both tissue types are clearly distinguishable. Simple modelling of the cell layers on the transistor allows extraction of a resistance term (Rc). OECT monitoring shows that barrier tissue cells lose their barrier function in a standard calcium switch assay, but remain adhered to the surface. Re-addition of calcium results in recovery of barrier tissue function. The entire process is continuously followed both electronically and optically. Finally, high resolution fluorescence imaging of live cells labelled with a red fluorescent actin marker demonstrates the versatility of this method for tracking molecular events optically, with direct correlation to electronic readouts.

[Strategies for bacterial virulence genes identification]. / Stratégies pour la recherche de gènes de virulence chez les bactéries pathogènes.

One of the recent preoccupations of medical microbiology has been to characterise the mechanisms of virulence of bacterial pathogens at the molecular level. One hundred years after Koch, Stanley Falkow proposed a new, molecular version of Koch's postulates to define avirulence gene: (a) the gene confers a certain phenotype to the studied bacteria, (b) inactivation of the gene abolishes the phenotype, (c)reintroduction of the gene restores the wild type to the mutant. Although this strategy, based upon mutagenesis and the use of experimental models, allows the identification of many genes, it is not comprehensive. Other methods can be used to complete the identification of virulence factors such as differential expression, either at the level of transcription (transcriptome) or at the level of protein expression (proteome). All these techniques are now supported by the data from complete genome sequencing projects. The pool of information obtained from these approaches allows the definition of the 'virulome', which is the assembly of factors a pathogen requires for virulence. Understanding the virulome will open the way to the development of new strategies for vaccination or the development of new generation of antimicrobials.

Chemo-enzymatic synthesis of the Galili epitope Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc on a homogeneously soluble PEG polymer by a multi-enzyme system.

The alpha-Gal trisaccharide Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc 11 was synthesized on a homogeneously soluble polymeric support (polyethylene glycol, PEG) by use of a multi-enzyme system consisting of beta-1,4-galactosyltransferase (EC 2.4.1.38), alpha-1,3-galactosyltransferase (EC 2.4.1.151), sucrose synthase (EC 2.4.1.13) and UDP-glucose-4-epimerase (EC 5.1.3.2). In addition workup was simplified by use of dia-ultrafiltration. Thus the advantages of classic chemistry/enzymology and solid-phase synthesis could be united in one. Subsequent hydrogenolytic cleavage afforded the free alpha-Gal trisaccharide.

Aromatic compound-dependent Brucella suis is attenuated in both cultured cells and mouse models.

The aroC gene of the facultative intracellular pathogen Brucella suis was cloned and sequenced. The cloned aroC gene complements Escherichia coli and Salmonella enterica serovar Typhimurium aroC mutants. A B. suis aroC mutant was found to be unable to grow in a defined medium without aromatic compounds. The mutant was highly attenuated in tissue culture (THP1 macrophages and HeLa cells) and murine virulence models.

Reduction of selenite and detoxification of elemental selenium by the phototrophic bacterium Rhodospirillum rubrum.

The effect of selenite on growth kinetics, the ability of cultures to reduce selenite, and the mechanism of detoxification of selenium were investigated by using Rhodospirillum rubrum. Anoxic photosynthetic cultures were able to completely reduce as much as 1. 5 mM selenite, whereas in aerobic cultures a 0.5 mM selenite concentration was only reduced to about 0.375 mM. The presence of selenite in the culture medium strongly affected cell division. In the presence of a selenite concentration of 1.5 mM cultures reached final cell densities that were only about 15% of the control final cell density. The cell density remained nearly constant during the stationary phase for all of the selenite concentrations tested, showing that the cells were not severely damaged by the presence of selenite or elemental selenium. Particles containing elemental selenium were observed in the cytoplasm, which led to an increase in the buoyant density of the cells. Interestingly, the change in the buoyant density was reversed after selenite reduction was complete; the buoyant density of the cells returned to the buoyant density of the control cells. This demonstrated that R. rubrum expels elemental selenium across the plasma membrane and the cell wall. Accordingly, electron-dense particles were more numerous in the cells during the reduction phase than after the reduction phase.

A homologue of the Agrobacterium tumefaciens VirB and Bordetella pertussis Ptl type IV secretion systems is essential for intracellular survival of Brucella suis.

Analysis of a TnblaM mutant of Brucella suis 1330, identified as being unable to multiply in Hela cells, allowed us to identify a 11 860 bp region of the B. suis genome encoding a type IV secretion system, homologous to the VirB system of Agrobacterium tumefaciens and the Ptl system of Bordetella pertussis. DNA sequence revealed 12 open reading frames (ORFs) encoding homologues of the 11 VirB proteins present in the pTi plasmid of Agrobacterium with a similar genetic organization, and a twelfth ORF encoding a putative lipoprotein, homologous to a protein involved in mating pair formation during bacterial conjugation and to adhesins used by Pseudomonas species to bind to plant roots. Phylogenetic trees based on the sequences of VirB4 and VirB9 protein homologues suggest that evolution of the systems from DNA transfer towards protein secretion did not stem from a single event but that the protein secretion systems have evolved independently. Four independent mutants in virB5, virB9 or virB10 were highly attenuated in an in vitro infection model with human macrophages. The virulence was restored by complementation with a plasmid containing the full virB region. The virB region appears to be essential for the intracellular survival and multiplication of B. suis.

[Perspectives and hopes in infectious pathology]. / Nouveautés et espoirs en pathologie infectieuse.

Over the last few decades, changes in socio-economic conditions and social practices as well as aggressive therapy of many diseases have led to the emergence of new infectious pathologies. These new pathologies are either associated with newly identified microbial species or the emergence of known microbes which have encountered new environments in which they are able to cause disease. Recent progress has allowed us to understand the mechanisms by which these pathogens express their virulence and will certainly allow us to diagnose and treat these infections more efficiently in the future.

Unconventional genomic organization in the alpha subgroup of the Proteobacteria.

Pulsed-field gel electrophoresis was used to analyze the genomic organization of 16 bacteria belonging or related to the family Rhizobiaceae of the alpha subgroup of the class Proteobacteria. The number and sizes of replicons were determined by separating nondigested DNA. Hybridization of an rrn gene probe was used to distinguish between chromosomes and plasmids. Members of the genus Agrobacterium all possess two chromosomes, and each biovar has a specific genome size. As previously demonstrated for Agrobacterium tumefaciens C58, the smaller chromosomes of Agrobacterium biovar 1 and Agrobacterium rubi strains appear to be linear. The genomes of Rhizobium strains were all of similar sizes but were seen to contain either one, two, or three megareplicons. Only one chromosome was present in the member of the related genus Phyllobacterium. We found one or two chromosomes in Rhodobacter and Brucella species, two chromosomes in Ochrobactrum anthropi, and one chromosome in Mycoplana dimorpha and Bartonella quintana; all of these genera are related to the Rhizobiaceae. The presence of multiple chromosomes is discussed from a phylogenetic and taxonomic point of view.

Differences in chromosome number and genome rearrangements in the genus Brucella.

We have studied the genomic structure and constructed the SpeI, PacI and I-CeuI restriction maps of the four biovars of the pathogenic bacterium Brucella suis. B. suis biovar 1 has two chromosomes of 2.1 Mb and 1.15 Mb, similar to those of the other Brucella species: B. melitensis, B. abortus, B. ovis and B. neotomae. Two chromosomes were also observed in the genome of B. suis biovars 2 and 4, but with sizes of 1.85 Mb and 1.35 Mb, whereas only one chromosome with a size of 3.1 Mb was found in B. suis biovar 3. We show that the differences in chromosome size and number can be explained by rearrangements at chromosomal regions containing the three rrn genes. The location and orientation of these genes confirmed that these rearrangements are due to homologous recombination at the rrn loci. This observation allows us to propose a scheme for the evolution of the genus Brucella in which the two chromosome-containing strains can emerge from an hypothetical ancestor with a single chromosome, which is probably similar to that of B. suis biovar 3. As the genus Brucella is certainly monospecific, this is the first time that differences in chromosome number have been observed in strains of the same bacterial species.

Genome structure and phylogeny in the genus Brucella.

PacI and SpeI restriction maps were obtained for the two chromosomes of each of the six species of the genus Brucella: B. melitensis, B. abortus, B. suis, B. canis, B. ovis, and B. neotomae. Three complementary techniques were used: hybridization with the two replicons as probes, cross-hybridization of restriction fragments, and a new mapping method. For each type strain, a unique I-SceI site was introduced in each of the two replicons, and the location of SpeI sites was determined by linearization at the unique site, partial digestion, and end labeling of the fragments. The restriction and genetic maps of the six species were highly conserved. However, numerous small insertions or deletions, ranging from 1 to 34 kb, were observed by comparison with the map of the reference strain of the genus, B. melitensis 16M. A 21-kb Spel fragment specific to B. ovis was found in the small chromosome of this species. A 640-kb inversion was demonstrated in the B. abortus small chromosome. All of these data allowed the construction of a phylogenetic tree, which reflects the traditional phenetic classification of the genus.

Investigation of the Actinobacillus actinomycetemcomitans genome by pulsed field gel electrophoresis.

Pulsed field gel electrophoresis was used to investigate nineteen strains of Actinobacillus actinomycetemcomitans. The genome was found to contain a single chromosome whose size we estimate to be 2300 kb from the sum of restriction fragments generated with rare cutting endonucleases. We detected the presence of large plasmids with sizes ranging from 35 to 300 kb. In some strains, extrachromosomal elements constitute over 20% of the total genome. Comparison of the profiles of ApaI digests of the 19 strains showed a high degree of polymorphism with 13 different profiles, providing a new tool for epidemiological studies.

Response of Brucella suis 1330 and B. canis RM6/66 to growth at acid pH and induction of an adaptive acid tolerance response.

Acid pH is an environmental stress often encountered by Brucella during both the "environmental" and the "pathogenic" stages of its life. We have investigated the behaviour of B. suis biovar 1 and B. canis in acid conditions. Growth at suboptimal pH was characterized by a dramatic reduction in growth yield due to an early onset of stationary phase. B. suis was more resistant to low pH than B. canis, which lysed at pH 4.6. Viable counts measured after a 4-h acid shock at pH 3.2 showed that the relative survival of B. suis was 1,000-fold greater than that of B. canis. An adaptive acid tolerance response (ATR) was induced in both species by culture at pH 5.8; however, while the acid-sensitive B. canis had more than a 2,000-fold increase in survival following acid shock at pH 3.2, the increase in survival of B. suis was only around 50-fold. The kinetics of the induction of ATR were followed: for B. suis, 1-2 h (1 generation) at pH 5.8 were required to induce acid tolerance (50-fold protection), and these levels remained constant over 24 h. B. canis became relatively acid-resistant after only 30-min exposure to pH 5.8. Levels of acid tolerance continued to increase and were maximal at 24 h. Stationary phase pH 7.2 cultures of either species did not exhibit acid resistance, suggesting that, like Salmonella, Brucella does not have an rpoS-controlled stationary phase acid resistance.

[Enzyme determination in vitro of inhibitor activity of beta-lactamase from tazobactam. Comparison with high performance liquid chromatography]. / Dosage enzymatique in vitro de l'activité inhibitrice de beta-lactamase du tazobactam. Comparaison à la chromatographic liquide à haute performance.

We developed an enzymatic method using nitrocefin to assay tazobactam in vitro. Tazobactam was incubated with TEM-1 beta-lactamase. Then, residual beta-lactamase activity was assayed by adding nitrocefin. This activity corresponded indirectly to the initial concentration of tazobactam. Within-assay, between-assay and accuracy coefficients of variation were below 15%. The correlation coefficients between enzymatic method and high performance liquid chromatography (the reference method)was 0.98. The enzymatic method is rapid, easy to perform and should be applied to daily clinical practice.

[A genetic and molecular biological study of the pathogenicity factors in Brucella]. / Geneticheskoe i molekuliarno-biologicheskoe izuchenie faktorov patogennosti brutsell.

The use of gene engineering techniques made it possible to obtain strain GSE830, capable of a higher level of expression of the gene of 38-kD protein in immunoblotting with sheep and rabbit antibrucellar sera in comparison with the expression of this gene of other Escherichia coli strains, containing recombinant plasmids with this gene. Due to the presence of the gene of 38-kD protein, recombinant E.coli strains were capable of survival in macrophage-like cell line U937 3.6-6.3 times more effectively. The model of interaction of Brucella pathogenic and nonpathogenic species with HeLa cells was studied. The bank of insertion mutants of B.suis virulent strain 1330 was studied with the use of transposon TnblaM. Out of 380 insertion mutants, 7 clones expressing beta-lactamase and having decreased capacity for multiplication in HeLa cells 48 hours after inoculation were selected. Detailed analysis revealed that 3 of them had lower adhesive capacity, 1 of them had lower invasive capacity and 3 other mutants were less capable of intracellular multiplication in HeLa cells than the initial B.suis strain 1330. All these 7 mutants had different sites of TnblaM insertion into the chromosome of B.suis strain 1330.

Polymorphism in Brucella strains detected by studying distribution of two short repetitive DNA elements.

Thirty-four Brucella reference or field strains representing all the species and biovars were studied by repetitive element sequence-based PCR, a PCR using primers complementary to two enterobacterial short repetitive sequences: repetitive extragenic palindromic and enterobacterial repetitive intergenic consensus sequences. All the stains showed a positive amplification, suggesting that the Brucella genome contains such sequences. Repetitive extragenic palindromic PCR was less discriminating than enterobacterial repetitive intergenic consensus PCR in terms of distinguishing strains, but a combination of the two methods was able to distinguish all the isolates, except for some strains belonging to biovars 3 and 9 of Brucella abortus. Repetitive element sequence-based PCR appears to be a simple and useful method for the study of brucellosis epidemiology.

In-vitro evaluation of beta-lactamase inhibition by latamoxef and imipenem.

The in-vitro 50% inhibitory concentrations (IC50s) of latamoxef and imipenem against a set of plasmid-mediated beta-lactamases including TEM-1, TEM-3, TEM-5 and TEM-10 were determined by an enzymatic method using nitrocefin as substrate. The IC50s of both antibiotics against extended-spectrum beta-lactamases were below 0.2 mg/L. The conventional spectrum beta-lactamase TEM-1 was not inhibited by either antibiotic at the highest concentration tested. Except for TEM-10 for which the IC50s of the two antibiotics were the same, imipenem showed significantly greater activity than latamoxef against TEM-3 and TEM-5. Clavulanic acid taken as a control demonstrated greater and wider inhibitory activity, but on the other hand it has no significant antibiotic activity.

Study of the organization of the genomes of Escherichia coli, Brucella melitensis and Agrobacterium tumefaciens by insertion of a unique restriction site.

Tn5Map, a Tn5 derivative containing the 18 bp I-SceI site, was delivered from a RP4-mobilizable, RK6-derived suicide vector to Escherichia coli HB101, Brucella melitensis and Agrobacterium tumefaciens C58, which all lack natural I-SceI sites in their genomes. Digestion of the DNA from Tn5Map-containing strains and analysis by pulsed-field gel electrophoresis (PFGE) revealed that these derivatives contained a single transposon insertion. These digests also gave direct and independent proof for the single circular chromosome of E. coli, and for the presence of two circular chromosomes in B. melitensis and of a circular and a linear chromosome in A. tumefaciens C58 (which also contains two large circular plasmids). This rapid and versatile technique is potentially applicable to the study of the genomic organization in all Gram-negative bacteria which support Tn5 transposition. Moreover, linearization of circular replicons could be the first step for a rapid method of physical mapping.

An enzymatic method for assaying sulbactam in human serum: comparison with high performance liquid chromatography.

An enzymatic method using nitrocefin as substrate was developed to assay sulbactam in human serum. Serum containing sulbactam was incubated with purified titrated TEM-1 beta-lactamase and nitrocefin was then added to the mixture to determine the remaining beta-lactamase activity and consequently the concentration of sulbactam. Assays were carried out on five patients with pulmonary infections receiving sulbactam plus amoxycillin iv. The values for serum sulbactam concentrations determined by the enzymatic method were compared with those determined by high performance liquid chromatography (HPLC). The correlation coefficient was 0.990 for serum sulbactam concentrations below 15 mg/L.