Antibacterial properties and reduction of MRSA biofilm with a dressing combining polyabsorbent fibres and a silver matrix.

OBJECTIVE: This study was designed to evaluate the antibacterial activity of a wound dressing which combines polyacrylate fibres and a silver lipido-colloid matrix (UrgoClean Ag, silver polyabsorbent dressing), against biofilm of methicillin-resistant Staphylococcus aureus (MRSA). METHOD: Samples of silver polyabsorbent dressing and the neutral form of this dressing (UrgoClean) were applied to biofilms of MRSA formed on a collagen I-coated surface, cultured for 24 hours. Different exposure times were tested (1, 2, 4 and 7 days) without dressing change. The biofilm reduction was quantified by using culture methods and by confocal laser scanning microscopy experiments. RESULTS: The application of the silver polyabsorbent dressing resulted in a significant decrease of the biofilm population by a log reduction of 4.6, after 24 hours of exposure. Moreover, the antibiofilm activity was maintained for 7 days with reduction values up to 4 log (reduction of biofilm superior to 99.99%). The application of the neutral dressing also induced a significant reduction of the concentration of sessile cells after 1 day (about 0.90 log). The results obtained with this neutral form of the dressing showed that the polyacrylate fibres were able to exert a mechanical disruption of the biofilm architecture. CONCLUSION: These in vitro experiments demonstrated that silver polyabsorbent dressing was able to strongly reduce the biofilm of MRSA. The antibiofilm mechanism of this dressing can be explained by a dual action of the polyabsorbent fibres (based on ammonium polyacrylate polymer around an acrylic core) which induced a mechanical disruption of the biofilm matrix and/or a sequestration of sessile cells, and the diffusion of silver ions which produced bactericidal activity. DECLARATION OF INTEREST: This study was supported by Laboratoires Urgo (Dijon). P. Janod is an employee of Laboratoires Urgo. The company had no influence on the experimental design and the interpretation of the results.

CD4+ T cell tolerance to parenchymal self-antigens requires presentation by bone marrow-derived antigen-presenting cells.

T cell tolerance to parenchymal self-antigens is thought to be induced by encounter of the T cell with its cognate peptide-major histocompatibility complex (MHC) ligand expressed on the parenchymal cell, which lacks appropriate costimulatory function. We have used a model system in which naive T cell receptor (TCR) transgenic hemagglutinin (HA)-specific CD4+ T cells are adoptively transferred into mice expressing HA as a self-antigen on parenchymal cells. After transfer, HA-specific T cells develop a phenotype indicative of TCR engagement and are rendered functionally tolerant. However, T cell tolerance is not induced by peptide-MHC complexes expressed on parenchymal cells. Rather, tolerance induction requires that HA is presented by bone marrow (BM)-derived cells. These results indicate that tolerance induction to parenchymal self-antigens requires transfer to a BM-derived antigen-presenting cell that presents it to T cells in a tolerogenic fashion.

Sensitivity to acetic acid, ability to colonize abiotic surfaces and virulence potential of Listeria monocytogenes EGD-e after incubation on parsley leaves.

AIM: To investigate how the survival of Listeria monocytogenes on parsley leaves may affect its ability to sustain process-related harsh conditions and its virulence. METHODS AND RESULTS: Parsley seedlings were spot inoculated with stationary phase cells of L. monocytogenes EGD-e and incubated for 15 days. Each day, bacterial cells were harvested and enumerated, and their ability to survive acetic acid challenge (90 min, pH 4.0), to colonize abiotic surfaces and to grow as biofilms was assessed. After a 3-log decrease over the first 48 h, the population stabilized to about 10(6) CFU g(-1) until the sixth day. After the sixth day, L. monocytogenes was no longer detected, even after specific enrichment. Incubation on parsley leaves affected the ability of L. monocytogenes to survive acetic acid challenge (90 min, pH 4.0) and to adhere to stainless steel although the ability to grow as biofilm was preserved. To further investigate these physiological alterations, the mRNA levels of six target genes (bsh, clpC, groEL, inlA, opuC, prfA) was quantified using reverse transcription qPCR after 5 h of incubation on parsley leaves. A decrease was observed in all but one (bsh) target, including groEL and clpC which are involved in resistance to salt and acid. Moreover, the decrease in the levels of inlA, prfA and opuC transcripts after incubation on parsley suggested a repression of some genes involved in pathogenicity. In vitro assessment of mammalian cell adherence and invasion using Caco-2 cells confirmed the repression of the virulence factor InlA; however, the virulence potential in vivo in the chick embryo model was not affected. CONCLUSION: Listeria monocytogenes did undergo rapid changes to adapt its physiology to the phyllosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the physiological changes undergone by L. monocytogenes during/after survival on parsley leaves.

Assessment of survival of Listeria monocytogenes, Salmonella Infantis and Enterococcus faecalis artificially inoculated into experimental waste or compost.

AIMS: To evaluate survival of pathogenic strains, Listeria monocytogenes and Salmonella Infantis and a sanitation indicator Enterococcus faecalis in composts at different stages of the composting process and during storage. METHODS AND RESULTS: The studied pathogenic and indicator strains, originally isolated from compost, were inoculated into compost samples from the various stages of the composting process. During incubation, indigenous microflora diversity was monitored with DGGE analysis. After 90 days of incubation, strain survival was observed in compost sampled before the beginning of the cooling phase, and DGGE analysis demonstrated an increase of microbial diversity up to the cooling phase. However, inoculated strains were not detected in composts after 30, 60 or 90 days of incubation in compost sampled after the start of the cooling phase. Microbial diversity also became stable, and DGGE profiles reached a maximum number of bands at this stage. CONCLUSIONS: Strain survival was not observed in stabilized composts. The cooling phase seems to be the turning point for pathogen survival and at this stage the indigenous microflora appeared to play a significant role in suppression. SIGNIFICANCE AND IMPACT OF THE STUDY: The importance of indigenous microflora in the survival of pathogens in four different composts was demonstrated. Stabilized composts were recommended for spreading on land.

Technological properties of Oenococcus oeni strains isolated from typical southern Italian wines.

AIMS: To isolate indigenous Oenococcus oeni strains suitable as starters for malolactic fermentation (MLF), using a reliable polyphasic approach. METHODS AND RESULTS: Oenococcus oeni strains were isolated from Nero di Troia wines undergoing spontaneous MLF. Samples were taken at the end of alcoholic fermentation and during MLF. Wine samples were diluted in a sterile physiological solution and plated on MRS and on modified FT80. Identification of O. oeni strains was performed by a polymerase chain reaction (PCR) experiment using strain-specific primers. Strains were further grouped using a multiplex RAPD-PCR analysis. Then, six strains were inoculated in two winelike media with two different ethanol concentrations (11 and 13% vol / vol) with a view to evaluate their capacity to grow and to perform MLF. In addition, a quantitative PCR (qRT-PCR) approach was adapted to monitor the physiological state of the strains selected. CONCLUSION: A positive correlation between the malolactic activity performance and the ability to develop and tolerate stress conditions was observed for two selected O. oeni strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The results reported are useful for the selection of indigenous MLF starter cultures with desired oenological traits from typical regional wines. It should be the base for the improvement in organoleptic quality of typical red wine.

Peptidases specific for proline-containing peptides and their unusual peptide-dependent regulation in Oenococcus oeni.

AIMS: Growth of the lactic acid bacterium (LAB) Oenococcus oeni, which is involved in malolactic fermentation during the winemaking process, is stimulated by peptides originating from yeast. In this study, we investigated the impact of peptides on O. oeni growth, peptidase activity and the expression of genes encoding the studied peptidases. METHODS AND RESULTS: Low levels of PepN activity and very high levels of PepI activity were observed in O. oeni, whereas levels of PepX activity were intermediate. The level of biosynthesis of these O. oeni peptidases was shown to depend on peptides present in the culture medium. These results were confirmed by transcriptional analyses of putative pep genes. The mechanism of repression by peptides did not involve a CodY-like regulator. CONCLUSIONS: Peptides from yeast decrease the levels of enzymatic activity and relative gene expression of O. oeni peptidases. Peptidases specific for proline-containing peptides are important for O. oeni nitrogen metabolism. SIGNIFICANCE AND IMPACT OF THE STUDY: We report here for the first time that the enzymes involved in the assimilation of proline-containing peptides by O. oeni differ from the well-described proteolytic system of milk LAB. This may reflect a specific adaptation to the wine environment.

Mutation of the oxaloacetate decarboxylase gene of Lactococcus lactis subsp. lactis impairs the growth during citrate metabolism.

AIMS: Citrate metabolism generates metabolic energy through the generation of a membrane potential and a pH gradient. The purpose of this work was to study the influence of oxaloacetate decarboxylase in citrate metabolism and intracellular pH maintenance in relation to acidic conditions. METHODS AND RESULTS: A Lactococcus lactis oxaloacetate decarboxylase mutant [ILCitM (pFL3)] was constructed by double homologous recombination. During culture with citrate, and whatever the initial pH, the growth rate of the mutant was lower. In addition, the production of diacetyl and acetoin was altered in the mutant strain. However, our results indicated no relationship with a change in the maintenance of intracellular pH. Experiments performed on resting cells clearly showed that oxaloacetate accumulated temporarily in the supernatant of the mutant. This accumulation could be involved in the perturbations observed during citrate metabolism, as the addition of oxaloacetate in M17 medium inhibited the growth of L. lactis. CONCLUSIONS: The mutation of oxaloacetate decarboxylase perturbed citrate metabolism and reduced the benefits of its utilization during growth under acidic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study allows a better understanding of citrate metabolism and the role of oxaloacetate decarboxylase in the tolerance of lactic acid bacteria to acidic conditions.

Oligopeptide assimilation and transport by Oenococcus oeni.

AIMS: Oenococcus oeni is a slow-growing wine bacterium with a low growth yield. It thrives better on complex nitrogen sources than on free amino-acid medium. We aimed to characterize the oligopeptide use of this micro-organism. METHODS AND RESULTS: Several peptides of two to eight amino-acid residues were able to provide essential amino acids. The disappearance of various peptides from extracellular medium was assessed with whole cells. Initial rates of utilization varied with the peptide, and free amino acids were released into the medium. CONCLUSIONS: Oenococcus oeni was able to transport the oligopeptides with two to five amino-acid residues tested and to hydrolyse them further. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has clear implications for the relationship between wine nitrogen composition and the ability of O. oeni to cope with its environment.

Effects of tetracyclines on aldosterone- and insulin-mediated Na+ transport in the toad urinary bladder.

The effect of oxytetracycline and demethylchlortetracycline on aldosterone- and insulin-mediated Na+ transport (short-circuit current) were examined in toad urinary bladders mounted in modified Ussing chambers. Oxytetracycline had little or no effect on either basal or aldosterone-mediated Na+ transport. In contrast, demethylchlortetracycline markedly inhibited both basal and aldosterone-mediated Na+ transport. Furthermore, demethylchlortetracycline inhibited the aldosterone response significantly out of proportion to its effects on basal Na+ transport. Neither of the drugs had an effect on insulin-mediated Na+ transport. Consequently, the natriuresis observed in certain patients treated with demethylchlortetracyline may be related to drug-induced renal resistance to the effects of aldosterone.

Microbial aerobic and anaerobic degradation of acrylamide in sludge and water under environmental conditions--case study in a sand and gravel quarry.

Polyacrylamides (PAMs) are used in sand and gravel quarries as water purification flocculants for recycling process water in a recycling loop system where the flocculants remove fine particles in the form of sludge. The PAM-based flocculants, however, contain residual amounts of acrylamide (AMD) that did not react during the polymerization process. This acrylamide is released into the environment when the sludge is discharged into a settling basin. Here, we explore the microbial diversity and the potential for AMD biodegradation in water and sludge samples collected in a quarry site submitted to low AMD concentrations. The microbial diversity, analyzed by culture-dependent methods and the denaturing gradient gel electrophoresis approach, reveals the presence of Proteobacteria, Cyanobacteria, and Actinobacteria, among which some species are known to have an AMD biodegradation activity. Results also show that the two main parts of the water recycling loop-the washing process and the settling basin-display significantly different bacterial profiles. The exposure time with residual AMD could, thus, be one of the parameters that lead to a selection of specific bacterial species. AMD degradation experiments with 0.5 g L(-1) AMD showed a high potential for biodegradation in all parts of the washing process, except the make-up water. The AMD biodegradation potential in samples collected from the washing process and settling basin was also analyzed taking into account on-site conditions: low (12 °C) and high (25 °C) temperatures reflecting the winter and summer seasons, and AMD concentrations of 50 µg L(-1). Batch tests showed rapid (as little as 18 h) AMD biodegradation under aerobic and anaerobic conditions at both the winter and summer temperatures, although there was a greater lag time before activity started with the AMD biodegradation at 12 °C. This study, thus, demonstrates that bacteria present in sludge and water samples exert an in situ and rapid biodegradation of AMD at low concentration, whatever the season, and in both the aerobic and anaerobic parts of the water recycling system.

Secretion of extracellular proteins by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a bacterial species of commercial value secreting numerous extracellular proteins, involved in pathogenesis. Most strains produce at least a lipase, a phospholipase, an alkaline phosphatase, an exotoxin and 2 proteases (elastase and alkaline protease). Various mechanisms for secretion of exoproteins appear to exist in P aeruginosa. Genetic analysis has led to the identification of 2 secretion pathways: i) a "general" secretion pathway, defined by the xcp mutations, which mediates secretion of most extracellular proteins, and; ii) an independent secretion pathway specific for alkaline protease. Our present knowledge on the pathways and components of the secretion machinery in P aeruginosa is reviewed in this article.

A bacterial toxicity assay performed with microplates, microluminometry and Microtox reagent.

We have developed a procedure for undertaking a Microtox-based test by coupling microplate and microluminometric technologies. Sample dilutions are prepared in a 96-well polystyrene microplate kept at 15 degrees C, while the Microtox reagent and diluent are placed in an opaque, microluminometry-compatible 96-well microplate also kept at 15 degrees C. Exposure begins when sample aliquots are brought into contact with bacterial reagents in the opaque microplate. After specific exposure times (5, 15, 30 and 60 min), bacterial luminescence is rapidly measured by placing the opaque microplate in a microluminometer. Reproducibility of the procedure, as well as general agreement of EC50 end-point values with published reports, is demonstrated herein after toxicity trials with six metals (Ni2+, Cd2+, Zn2+, Pb2+, Cu2+ and Cr6+). Results suggest that a 60-min exposure time may have value in getting more "sensitivity mileage" out of this Microtox-based assay. This microplate procedure possesses attractive features that augment sample throughput and information output. Further refinement and validation studies are ongoing in our laboratories.

Effect of estrogen replacement therapy on corrected thrombolysis in myocardial infarction frame count.

The effect of chronic estrogen replacement therapy on the corrected Thrombolysis In Myocardial Infarction trial frame count of the left anterior descending coronary artery was assessed in 122 postmenopausal women. With use of multivariate analysis to account for confounding variables likely to affect the corrected Thrombolysis In Myocardial Infarction trial frame count, no chronic effect of estrogen replacement therapy on coronary blood flow was documented.

Preoperative factors predisposing to early postoperative atrial fibrillation after isolated coronary artery bypass grafting.

An analysis of 183 patients in sinus rhythm who underwent coronary artery bypass grafting was conducted to determine the association of multiple preoperative factors, including an elevated left ventricular end-diastolic pressure, with early postoperative atrial fibrillation. An association with advanced age, a history of atrial fibrillation, and preoperative digoxin use was found, but not with an elevated left ventricular end-diastolic pressure, irrespective of left ventricular systolic function.

The main cold shock protein of Listeria monocytogenes belongs to the family of ferritin-like proteins.

The transfer of the food-borne pathogen Listeria monocytogenes from 30 to 5 degrees C was characterized by the sharp induction of a low molecular mass protein. This major cold shock protein has an isoelectric point at pH 5.1 and a molecular mass of about 18 kDa, as observed on two-dimensional gel electrophoresis (2-DE) pattern. Its N-terminal sequence, obtained from the 2-DE spot, shared a complete sequence identity with a Listeria innocua non-heme iron-binding ferritin. The purification of these ferritin-like proteins (Flp) revealed a native molecular mass of about 100-110 kDa which indicates a polypeptide composed of six 18 kDa-subunits. Northern analysis indicated the presence of a 0.8-kb monocistronic mRNA in exponential growing cells and an important increase inflp mRNA amount after a downshift but also an upshift in temperature.

Cloning and expression in Escherichia coli of a phoA gene encoding a phosphate-irrepressible alkaline phosphatase of Zymomonas mobilis.

The Zymomonas mobilis phoA gene, encoding a phosphate-irrepressible alkaline phosphatase (ZAPase), was cloned and its expression was studied in phoA mutants of Escherichia coli. The ZAPase was recovered in the soluble fraction of E. coli. The enzyme was synthesized constitutively and its synthesis not repressed by phosphate, unlike the phoA gene of E. coli. The phoA gene of Z. mobilis was mutagenized by Mini Mu PR13 and the mutated gene crossed into Z. mobilis in order to obtain phoA mutants by reverse genetics. Although Z. mobilis mutants with Mini Mu PR13 integrated in the chromosome were obtained, none had an allele replacement for none was defective in ZAPase.

Cloning and sequencing of the gene encoding alpha-acetolactate decarboxylase from Leuconostoc oenos.

The alsD gene encoding alpha-acetolactate decarboxylase was isolated from a genomic library of Leuconostoc oenos, using a screening procedure developed on microtiter plates. The nucleotide sequence of alsD encodes a putative protein of 239 amino acids showing significant similarity with other bacterial alpha-acetolactate decarboxylases. Upstream from alsD lies an open reading frame (alsS) which is highly similar to bacterial genes coding for catabolic alpha-acetolactate synthases. Northern (RNA) blotting analyses indicated the presence of a 2.4-kb dicistronic transcript of alsS and alsD. This suggests that the alsS and alsD genes are organized in a single operon.

Acid sensitivity of neomycin-resistant mutants of Oenococcus oeni: a relationship between reduction of ATPase activity and lack of malolactic activity.

Mutants of Oenococcus oeni were isolated as spontaneous neomycin-resistant mutants. Three of these mutants harbored a significantly reduced ATPase activity that represented 50% of that of the wild-type strain. Their growth rates were also impaired at pH 5.3 (46-86% of the wild-type level). However, the profiles of sugar consumption appeared identical to those of the parental strain. At pH 3.2, all the mutant strains failed to grow and a drastic decrease in viability was observed after an acid shock. Surprisingly, all the isolated mutants were devoid of malolactic activity. These results suggest that the ATPase and malolactic activities of O. oeni are linked to each other and play a crucial role in the mechanism of resistance to an acid stress.

Characterization of the effects of aluminum on luciferase biosensors for the detection of ecotoxicity.

Luciferase-based biosensors are becoming increasingly used for environmental monitoring. A transcriptional fusion of the Vibrio harveyi luxAB genes (encoding bacterial luciferase) to the fliC gene of Escherichia coli was constructed and luminescence shown to be induced (in liquid media) in the presence of 1-10 micrograms/ml aluminum, but not copper, iron or nickel. Moreover, luminescence is markedly increased at pH 5.5, where aluminum is more soluble than at pH 7.0. However, aluminum also stimulated luciferase activity when the luxAB genes were located in the xyl operon. This suggests that aluminum stimulates luciferase enzyme activity in vivo. These results are specific to E. coli, as no such aluminum stimulation was observed in the luminescent bacterium V. harveyi. These results have important implications in the generalized use of these clones for environmental monitoring, where aluminum can be present at elevated concentrations.

Regulation of stress response in Oenococcus oeni as a function of environmental changes and growth phase.

Oenococcus oeni is a lactic acid bacterium which is able to grow in wine and perform malolactic fermentation. To survive and grow in such a harsh environment as wine, O. oeni uses several mechanisms of resistance including stress protein synthesis. The molecular characterisation of three stress genes hsp18, clpX, trxA encoding for a small heat shock protein, an ATPase regulation component of ClpP protease and a thioredoxin, respectively, allow us to suggest the existence in O. oeni of multiple regulation mechanisms as is the case in Bacillus subtilis. One common feature of these genes is that they are expressed under the control of housekeeping promoters. The expression of these genes as a function of growth is significantly different. Surprisingly, the clpX gene, which is induced by heat shock, was highly expressed in the early phase of growth. In addition to stress protein synthesis, adaptation to the acid pH of wine requires efficient cellular systems to extrude protons. Using inhibitors specific for different types of ATPases, we demonstrated the existence of H+-ATPase and P-type ATPase.