Effects of a pulsed light-induced stress on Enterococcus faecalis.

AIMS: Pulsed light (PL) technology is a surface decontamination process that can be used on food, packaging or water. PL efficiency may be limited by its low degree of penetration or because of a shadow effect. In these cases, surviving bacteria will be able to perceive PL as a stress. Such a stress was mimicked using low transmitted energy conditions, and its effects were investigated on the highly environmental adaptable bacterium Enterococcus faecalis V583. METHODS AND RESULTS: In these laboratory conditions, a complete decontamination of the artificially inoculated medium was performed using energy doses as low as 1.8 J cm(-2) , while a treatment of 0.5, 1 and 1.2 J cm(-2) led to a 2.2, 6 and 7-log(10) CFU ml(-1) reduction in the initial bacterial population, respectively. Application of a 0.5 J cm(-2) pretreatment allowed the bacteria to resist more efficiently a 1.2 J cm(-2) subsequent PL dose. This 0.5 J cm(-2) treatment increased the bacterial mutation frequency and affected the abundance of 19 proteins as revealed by a global proteome analysis. CONCLUSIONS: Enterococcus faecalis is able to adapt to a PL treatment, providing a molecular response to low-energy PL dose, leading to enhanced resistance to a subsequent treatment and increasing the mutation frequency. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives further insights on Ent. faecalis capacities to adapt and to resist to stress.

In vitro evaluation of the probiotic potential of Lactobacillus salivarius SMXD51.

Lactobacillus salivarius SMXD51 was previously isolated from the cecum of a Tunisian poultry and found to produce a bacteriocin-like substance highly active against the foodborne pathogen Campylobacter jejuni. The aim of this study was to examine some probiotic properties of the strain: acid and bile tolerance, capacity of adhesion, stimulation of immune defences (IL-6, IL-8, IL-10 and ß-defensin 2), and modulation of the barrier integrity. The results showed that L. salivarius SMXD51 can tolerate gastrointestinal conditions (acid and bile), adhere to intestinal cells and stimulate the immune system. The bacterium strengthened the intestinal barrier functions through the increase of the transepithelial electrical resistance (TEER) and reinforcement of the F-actin cytoskeleton. One hour pretreatment with L. salivarius SMXD51 protected against Pseudomonas aeruginosa PAO1-induced decrease of TEER and damage of the F-actin cytoskeleton. Our results highlight that L. salivarius SMXD51 fulfils the principle requirements of an efficient probiotic and may be seen as a reliable candidate for further validation studies in chicken.

Structural and functional evolution of the translocator protein (18 kDa).

Translocator proteins (TSPO) are the products of a family of genes that is evolutionarily conserved from bacteria to humans and expressed in most mammalian tissues and cells. Human TSPO (18 kDa) is expressed at high levels in steroid synthesizing endocrine tissues where it localizes to mitochondria and functions in the first step of steroid formation, the transport of cholesterol into the mitochondria. TSPO expression is elevated in cancerous tissues and during tissue injury, which has lead to the hypothesis that TSPO has roles in apoptosis and the maintenance of mitochondrial integrity. We recently identified a new paralog of Tspo in both the human and mouse. This paralog arose from an ancient gene duplication event before the divergence of the classes aves and mammals, and appears to have specialized tissue-, cell-, and organelle-specific functions. Evidence from the study of TSPO homologs in mammals, bacteria, and plants supports the conclusion that the TSPO family of proteins regulates specialized functions related to oxygen-mediated metabolism. In this review, we provide a comprehensive overview of the divergent function and evolutionary origin of Tspo genes in Bacteria, Archaea, and Eukarya domains.

Adaptation of Pseudomonas aeruginosa to a pulsed light-induced stress.

AIMS: Pulsed light (PL) technology is an efficient surface decontamination process. Used in low transmitted energy conditions, PL induces a stress that can be perceived by bacteria. The effect of such a PL stress was investigated on the highly environmental adaptable germ Pseudomonas aeruginosa PAO1. METHODS AND RESULTS: Pulses of transmitted energy (fluence) reaching 1·8Jcm(-2) can kill 10(9) bacteria. Application of a lower sublethal PL dose allowed the bacteria to resist and survive more efficiently to a subsequent dose of PL. This sublethal dose was not increasing the mutation frequency of Ps. aeruginosa, but altered the abundance of 15 proteins as revealed by a global proteome analysis, including stress-induced proteins, phage-related proteins, energy and carbon metabolisms, cell motility, and transcription and translation regulators. CONCLUSIONS: A response to a low-energy PL dose takes place in Ps. aeruginosa, reducing the energy conversion systems, while increasing transcription and translation processes to produce proteins involved in chaperone mechanisms and phage-related proteins, probably to protect the bacterium against a new PL-induced stress. SIGNIFICANCE AND IMPACT OF THE STUDY: Taken together, these results suggest that a low-energy PL dose is sufficient to provoke adaptation of Ps. aeruginosa, leading to enhancing its resistance to a subsequent lethal treatment.

Toxicity induced by cumene hydroperoxide in PC12 cells: protective role of thiol donors.

Oxidative shock and production of reactive oxygen species are known to play a major role in situations leading to neuron degeneration, but the precise mechanisms responsible for cell degeneration remain uncertain. In the present article, we have studied in PC 12 cells the effect of cumene hydroxyperoxide on both cell metabolism and morphology. We observed that relatively low concentrations of the drug (100 µM) led to a significant decrease in the cellular content of ATP and reduced glutathione as well as to a decreased mitochondrial potential. These metabolic alterations were followed by an important increase in intracellular free calcium and membrane disruption and death. In parallel, we observed profound changes in cell morphology with a shortening of cell extensions, the formation of ruffles and blebs at the cell surface, and a progressive detachment of the cells from the surface of the culture flasks. We also showed that addition of thiol donors such as N-acetylcysteine or ß-mercaptoethanol, which were able to enhance cell glutathione content, almost completely protected PC 12 cells from the toxic action of cumene hydroperoxide whereas pretreatment by buthionine sulfoximine, a selective inhibitor of GSH synthesis, enhanced its action.

Phenotypic variation in the Pseudomonas fluorescens clinical strain MFN1032.

Pseudomonas fluorescens is a highly heterogeneous species and includes both avirulent strains and clinical strains involved in nosocomial infections. We previously demonstrated that clinical strain MFN1032 has hemolytic activity involving phospholipase C (PlcC) and biosurfactants (BSs), similar to that of the opportunistic pathogen Pseudomonas aeruginosa. When incubated under specific conditions, MFN1032 forms translucent phenotypic variant colonies defective in hemolysis, but not necessarily in PlcC. We analyzed eight variants of the original strain MFN1032 and found that they clustered into two groups. Mutations of genes encoding the two-component regulatory system GacS/GacA are responsible for phenotypic variation in the first group of variants. These group 1 variants did not produce secondary metabolites and had impaired biofilm formation. The second group was composed of hyperflagellated cells with enhanced biofilm capacity: they did not produce BSs and were thus unable to swarm. Artificial reduction of the intracellular level of c-di-GMP restored the ability to form biofilm to levels shown by the wild type, but production of BSs was still repressed. Phenotypic variation might increase the virulence potential of this strain.

Non-thermal plasma technologies: new tools for bio-decontamination.

Bacterial control and decontamination are crucial to industrial safety assessments. However, most recently developed materials are not compatible with standard heat sterilization treatments. Advanced oxidation processes, and particularly non-thermal plasmas, are emerging and promising technologies for sanitation because they are both efficient and cheap. The applications of non-thermal plasma to bacterial control remain poorly known for several reasons: this technique was not developed for biological applications and most of the literature is in the fields of physics and chemistry. Moreover, the diversity of the devices and complexity of the plasmas made any general evaluation of the potential of the technique difficult. Finally, no experimental equipment for non-thermal plasma sterilization is commercially available and reference articles for microbiologists are rare. The present review aims to give an overview of the principles of action and applications of plasma technologies in biodecontamination.

Comparative study of 7 fluorescent pseudomonad clinical isolates.

There is some debate about the potential survival of Pseudomonas fluorescens at temperatures above 37 degrees C and its consequences for infectious potential, owing to the heterogeneity of clinical strains. Seven clinical strains growing at 37 degrees C or more were submitted for polyphasic identification; 2 were identified as Pseudomonas mosselii and 4 were precisely characterized as P. fluorescens bv. I or II. The binding indexes on glial cells of the strains identified as P. fluorescens bv. I and P. mosselii were compared with that of a reference psychrotrophic strain, P. fluorescens MF37 (bv. V). Clinical P. fluorescens had a similar adherence potential range than strain MF37. Conversely, the binding indexes for P. mosselii strains were 3 times greater than that for strain MF37. These data, and those obtained by comparing the cytotoxic activities of P. fluorescens clinical strains, suggest the existence of different virulence mechanisms, leading either to a low infectious form or to a microorganism with cytotoxic activity in the same range as that of P. mosselii or even Pseudomonas aeruginosa.

Gliding arc discharge in the potato pathogen Erwinia carotovora subsp. atroseptica: mechanism of lethal action and effect on membrane-associated molecules.

Gliding arc (glidarc) discharge is a physicochemical technique for decontamination at atmospheric pressure and ambient temperature. It leads to the destruction of bacterial phytopathogens responsible for important losses in industrial agriculture, namely, Erwinia spp., without the formation of resistant forms. We investigated the effect of a novel optimized prototype allowing bacterial killing without lag time. This prototype also decreases the required duration of treatment by 50%. The study of the time course effect of the process on bacterial morphology suggests that glidarc induces major alterations of the bacterial membrane. We showed that glidarc causes the release of bacterial genomic DNA. By contrast, an apparent decrease in the level of extractible lipopolysaccharide was observed; however, no changes in the electrophoretic pattern and cytotoxic activity of the macromolecule were noted. Analysis of extractible proteins from the outer membrane of the bacteria revealed that glidarc discharge induces the release of these proteins from the lipid environment, but may also be responsible for protein dimerization and/or aggregation. This effect was not observed in secreted enzymatic proteins, such as pectate lyase. Analysis of the data supports the hypothesis that the plasma generated by glidarc discharge is acting essentially through oxidative mechanisms. Furthermore, these results indicate that, in addition to effectively destroying bacteria, glidarc discharge should be used to improve the extraction of bacterial molecules.

Lethal effect of the gliding arc discharges on Erwinia spp.

AIMS: To compare the decontamination performances of glidarc on strains of Erwinia of industrial interest. METHODS AND RESULTS: Cultures of Erwinia carotovora carotovora, Erwinia carotovora atroseptica and Erwinia chrysanthemi taken in stationary phase were exposed to the plasma generated by electric discharges in a gliding arc reactor prototype. The kinetics of destruction of bacteria were followed by direct platting. All bacterial strains presented a three-phase destruction kinetics leading to an apparent sterilization within 10 min. Epifluorescent observations using life/dead probes revealed the absence of viable but not cultivable resistant forms. Measurement of the physical parameters of the medium confirmed that the technique was nonthermal but that reactive species responsible for a decrease of the pH were generated. However, even after neutralization the medium did not allow bacterial growth. CONCLUSIONS: The results demonstrate that glidarc allows a rapid and complete destruction of planctonic strains of Erwinias without formation of resistant forms. SIGNIFICANCE AND IMPACT OF THE STUDY: The reduction rate obtained by this technique shows the great industrial interest of glidarc for decontamination and suggests that it can be used for sterilization of industrial water effluents.

Sequential activation of constitutive and inducible nitric oxide synthase (NOS) in rat cerebellar granule neurons by pseudomonas fluorescens and invasive behaviour of the bacteria.

Previous studies have shown that Pseudomonas fluorescens and its lipopolysaccharide (LPS) exert dose-related cytotoxic effects on neurons and glial cells. In the present work, we investigated the time course effect of P. fluorescens MF37 and its LPS on cultured rat cerebellar granule neurons. The kinetics of binding of P. fluorescens to cerebellar granule neurons is rapid and reaches a mean of 3 bacteria/cell after 5 h. As demonstrated by measurement of the concentration of nitrite in the culture medium, P. fluorescens induces a rapid stimulation (3 h) of the nitric oxide synthase (NOS) activity of the cells. In contrast, LPS extracted from P. fluorescens requires a long lag phase (24 h) before observation of an activation of NOS. Measurement of the membrane resting potential of granule neurons showed that within 3 h of incubation there was no difference of effect between the action of P. fluorescens and that of its endotoxin. Two complementary approaches allowed to demonstrate that P. fluorescens MF37 presents a rapid invasive behaviour suggesting a mobilisation of calcium in its early steps of action. The present study reveals that P. fluorescens induces the sequential activation of a constitutive calcium-dependent NOS and that of an inducible NOS activated by LPS. Our results also suggest that in P. fluorescens cytotoxicity and invasion are not mutually exclusive events.

Pseudomonas fluorescens as a potential pathogen: adherence to nerve cells.

In order to determine the infectious potential of the psychrotrophic bacterium Pseudomonas fluorescens, a species closely related to the opportunistic pathogen P. aeruginosa, we investigated the binding activity of this bacterium on primary cultures of rat neonate cortical neurons and glial cells, adrenal paraneurons and NG108-15 neuroblastoma cells. Incubated at concentrations of 10(6) and 10(8) CFU/mL, P. fluorescens MF37 exhibited a high binding activity on neurons in the same range as that of P. aeruginosa PAO1. A significant, but lower, adherence of P. fluorescens was also detected on glial cells and adrenal paraneurons. In contrast, when P. fluorescens MF37 or P. aeruginosa PAO1 were incubated with neuroblastoma cells, no binding was observed. In neurons, the association of P. fluorescens with the plasma membrane occurred both on neurites and cell body. Leakage of the cytoplasmic content was frequently noted. Studies performed using the fluorescent probe Hoechst 33258 revealed that in 10% of neurons, P. fluorescens induced the appearance of densely stained clusters of DNA that was typical of an early step of apoptosis. In glial cells exposed to P. fluorescens, marked changes in the morphology of the nucleus, including fragmentation into lobular structures and aggregation of DNA, were also reminiscent of the existence of a possible apoptotic mechanism. Taken together, these results reveal that P. fluorescens can bind to nerve cells and affect their physiology and, in agreement with recent clinical observations, suggest that P. fluorescens could behave as a pathogen.

Effect of the triakontatetraneuropeptide (TTN) on corticosteroid secretion by the frog adrenal gland.

Diazepam-binding inhibitor (DBI) was initially isolated from the rat brain as a result of its ability to compete with benzodiazepines for their receptors. Immunohistochemical studies have recently shown the presence of peripheral-type benzodiazepine receptor (PBR)- and DBI-like immunoreactivity in the frog adrenal gland. The aim of the present study was to investigate the effect of two biologically active DBI-derived peptides, the triakontatetraneuropeptide [TTN; DBI(17-50)] and the octadecaneuropeptide [ODN; DBI(33-50)], on corticosteroid secretion by frog adrenocortical cells. Exposure of frog adrenal explants to graded concentrations of TTN (3.16 x 10(-8) to 3.16 x 10(-6) M) induced a dose-related increase in corticosterone and aldosterone secretion. In contrast, ODN did not modify corticosteroid output. When repeated pulses of TTN (10(-6) M) were administered at 2-h intervals, the response of the adrenal explants to the second dose of TTN was markedly reduced, suggesting the existence of a desensitization phenomenon. Exposure of dispersed adrenal cells to TTN also induced a marked stimulation of corticosteroid secretion, indicating that TTN acts directly on adrenocortical cells. The central-type benzodiazepine receptor (CBR) agonist, clonazepam, did not stimulate corticosteroid secretion and the CBR antagonist, flumazenil, did not block the stimulatory action of TTN. Similarly, the PBR agonist, Ro5-4864, did not mimic the stimulatory effect of TTN and the PBR antagonist, flunitrazepam, did not affect the stimulatory action of TTN. The present study provides the first evidence for a stimulatory effect of TTN on intact adrenocortical cells. The receptor mediating the corticotropic action of TTN is not related to central- or peripheral-type benzodiazepine receptors. Our data suggest that TTN, released by chromaffin cells, may act as a paracrine factor regulating the activity of adrenocortical cells.

The endozepine triakontatetraneuropeptide diazepam-binding inhibitor [17-50] stimulates neurosteroid biosynthesis in the frog hypothalamus.

Neurons and glial cells are capable of synthesizing various bioactive steroids, but the neuronal mechanisms controlling neurosteroid-secreting cells are poorly understood. In the present study, we have investigated the possible effect of an endogenous ligand of benzodiazepine receptors, the triakontatetraneuropeptide [17-50] (TTN), on steroid biosynthesis in the frog hypothalamus. Immunohistochemical studies revealed that most hypothalamic neurons expressing 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase also contained peripheral-type benzodiazepine receptor-like immunoreactivity. Confocal laser scanning microscopic analysis revealed that the peripheral-type benzodiazepine receptor-immunoreactive material was located both in the cytoplasm and at the periphery of the cell bodies. By using the pulse-chase technique, TTN was found to stimulate the conversion of [3H]pregnenolone into various steroids, including 17-hydroxypregnenolone, 5 alpha-dihydrotestosterone and 17-hydroxyprogesterone, in a dose-dependent manner. The peripheral-type benzodiazepine receptor agonist Ro5-4864 mimicked the stimulatory effect of TTN on the formation of neurosteroids. The peripheral-type benzodiazepine receptor antagonist PK11195 significantly reduced the effect of TTN on neurosteroid synthesis, while the central-type benzodiazepine receptor antagonist flumazenil did not affect the formation of neurosteroids evoked by TTN. These data indicate that TTN stimulates the biosynthesis of 3-keto-17 alpha-hydroxysteroids in frog hypothalamic neurons through activation of peripheral-type benzodiazepine receptors likely located at the plasma membrane level.

Characterization of pars intermedia connections in amphibians by biocytin tract tracing and immunofluorescence aided by confocal microscopy.

Biocytin, recently introduced in neuroanatomical studies, was used as a retrograde tract tracer in combination with immunofluorescence in order to analyse the neurochemical characters of some central neuronal projections to the pars intermedia in two amphibian species, the anuran Rana esculenta and the urodele Triturus carnifex. After biocytin insertions in the pars intermedia, neurons became retrogradely labelled in the suprachiasmatic hypothalamus and the locus coeruleus of the brainstem in both species. Some scattered biocytin-labelled neurons were observed in the preoptic area. Moreover, working on the same sections, immunofluorescence revealed a number of codistributions and, in some cases, colocalization in the same neurons of biocytin labellings and immunopositivity for (1) tyrosine hydroxylase in the suprachiasmatic hypothalamus and the locus coeruleus of Rana and Triturus, (2) gamma-aminobutyric acid in the suprachiasmatic hypothalamus of Rana and Triturus and (3) neuropeptide Y in the suprachiasmatic hypothalamus of Rana. The specificity of such colocalizations was fully confirmed using dual-channel confocal laser scanning microscopy analysis.