Membrane proteomes of Pseudomonas aeruginosa and Acinetobacter baumannii.

Acinetobacter baumannii and Pseudomonas aeruginosa are known for their intrinsic resistance to antibiotics. Between mechanisms involved in this resistance, diminished expression of outer membrane proteins and up-regulation of efflux pumps play an important role. The characterization of membrane proteins is consequently necessary because of their importance in the antibiotic resistance but also in virulence. This review presents proteomic investigations aiming to describe the protein content of the membranes of these two bacterial species.

Long-term incomplete xylose fermentation, after glucose exhaustion, with Candida shehatae co-immobilized with Saccharomyces cerevisiae.

Saccharomyces cerevisiae and Candida shehatae were co-immobilized in an agar sheet which was introduced in an original two-chambered bioreactor asymmetrically fed in a batch mode with a mixture of glucose and xylose in a ratio of 35:15. The two sugars were consumed simultaneously. All glucose was fermented but only 20% of xylose. After incubation, yeast cells recovered from different areas of the agar sheet (close to, called Hi, and distant from, called Ho, the substrate chamber) were cultured as suspended cells in fresh culture medium provided with xylose or the sugar mixture. Xylose utilization by gel released Hi yeasts was significantly delayed compared to the Ho culture. Ethanol consumption by Hi yeasts in the two-substrate medium occurred after glucose exhaustion despite the presence of xylose. The waste medium resulting from incubation of the immobilized-cell structure inhibited xylose utilization by C. shehatae. Our results suggested that batch fermentation most probably favoured this incomplete xylose fermentation.

Occurrence of sessile Pseudomonas oryzihabitans from a karstified chalk aquifer.

Pseudomonas oryzihabitans is an uncommon pathogen that may cause opportunistic infections. Although it has been previously isolated from the environment, the source of human infection has not been well documented. In this study, we describe the presence of P. oryzihabitans adhering on suspended particulate matters recovered from karst groundwaters. The isolated pathogen was capable of forming biofilms on silicon supports and clay beads. Adherent P. oryzihabitans cells displayed a high resistance to chlorine as compared with the same organisms cultured in the planktonic mode. These results demonstrate that aquifer biofilms are potential environmental sources for water-born P. oryzihabitans infections and that bacterial attachment might affect drinking water purification.

Occurrence and phenotypic characterization of Yersinia ruckeri strains with biofilm-forming capacity in a rainbow trout farm.

The presence of Yersinia ruckeri in a French fish farm was investigated. Y. ruckeri was isolated mainly from algae and sediment samples rather than from water. Twenty-two Y. ruckeri isolates were obtained, and three strains were distinguished by enterobacterial repetitive intergenic consensus PCR amplification. These strains were able to adhere to solid supports. This characteristic was correlated with flagellum-mediated motility. Killing experiments showed that sessile cells were more resistant to oxolinic acid than their planktonic counterparts. Our results demonstrate that surface colonization of fish farm tanks by Y. ruckeri biofilms is a potential source of recurrent infection for extended periods of time.

Primary biodegradability of mineral base oils in relation to their chemical and physical characteristics.

The primary biodegradability of 32 mineral base (i.e., unformulated) oils of paraffinic nature was evaluated using the CEC L-33-A-93 test. These oils were refinery products obtained by varying manufacturing processes. Biodegradation percentages ranged between 15% and 75%, i.e., below the commonly accepted standards for environmentally-compatible lubricants. Biodegradability values were compared to the overall chemical composition and main physical properties of base oils. Biodegradability decreased with increasing levels of aromatic and/or polar compounds in the tested oils. For most oils, the biodegradation percentage increased with the viscosity index, but was a decreasing function of the kinematic viscosity (KV), the pour point, the flash point (FP) and the refractive index (RI). Linear relationships between biodegradability and FP or RI values were observed. These results show that, beside chemical features such as the contents in polar and aromatic compounds, simple physical magnitudes such as KV and RI, commonly used to characterize lubricant properties, may be useful parameters for predicting the biodegradability of mineral base oils.

Cell immobilization induces changes in the protein response of Escherichia coli K-12 to a cold shock.

We have compared the protein expression of gel-entrapped Escherichia coli cells submitted to a cold shock at 4 degrees C with those of exponential- and stationary-phase free-floating counterparts. Autoradiograms of two-dimensional gel electrophoresis patterns of proteins radiolabeled with L-[35S]methionine were compared using computing scanning densitometry. The levels of 203 proteins synthesized during the temperature shift were significantly and reproducibly higher than those corresponding to synthesis at 37 degrees C. A principal component analysis (PCA) was performed on the synthesis levels of these 203 proteins in the different incubation conditions tested. This study showed that the protein response of immobilized cells after the cold shock was significantly different from those of exponential- and stationary-phase free-floating organisms. For instance, protein SSB was specifically overexpressed by shocked immobilized organisms. Such induction of specific molecular mechanisms in immobilized bacteria might explain the high resistance of sessile-like organisms to stresses.

Sugar beet Ca-pectate gel as a cost-effective immobilized-cell matrix for metal biosorption.

Whole bacterial (Pseudomonas putida) and yeast (Saccharomyces cerevisiae) cells were immobilized by entrapment in gel beads of Ca-pectate obtained from sugar beet pulp and gel beads of commercial Ca-alginate (of algal origin). These immobilized-cell particles were tested for Cd2+ removal from dilute aqueous solutions and their mechanical properties were evaluated. Only Ca-pectate gel beads loaded with yeast cells displayed enhanced cadmium-binding efficiency (13.8 mg Cd2+ per g dry matter) as compared to sterile beads (10.7 mg/g). Sterile Ca-pectate gel beads were noticeably more brittle than algal alginate counterparts. The presence of cells reduced the mechanical resistance of both types of beads, however. The practical use of cell-loaded sugar beet Ca-pectate gel as a metal biosorbent is discussed in light of these results.

Molecular weight characteristics of alginate produced by Azotobacter vinelandii in a membrane bioreactor.

A membrane bioreactor which combines a continuous fermentor and a cross-flow microfiltration module enabling continuous cell recycling and removal of the fermentation products freed of microbial cells was tested for the production of alginate by Azotobacter vinelandii. The molecular weight characteristics of the polysaccharide produced were investigated. The macromolecular characteristics of alginate recovered by first filtering batch fermentation broths through different microporous ceramic membranes with pore sizes varying from 0.45 to 1.4 microm seemed to be independent of the MPS. The 0.8-microm membrane pore size (MPS) was selected for continuous alginate production in the membrane bioreactor. A mean yield value of 0.21 g alginate g(-1) saccharose was obtained and an increase in alginate polydispersity as incubation proceeded was observed, probably due to enzymatic degradation of the polysaccharide.

Substituting Coomassie Brilliant Blue for bromophenol blue in two-dimensional electrophoresis buffers improves the resolution of focusing patterns.

In a new area of postgenomics challenges, the optimization of protein identification has become a central goal in microbiochemistry. In this work, we demonstrate that the substitution of Coomassie Brilliant Blue for bromophenol blue in two-dimensional electrophoresis (2-DE) buffers improves the focusing of whole proteins from Pseudomonas aeruginosa. This improvement of focusing concerns more particularly basic proteins. This enhancement may be attributed to a better transfer from the first to the second dimension, which probably highlights an increase in the solubility of proteins in the IPG strips. Hence, the use of an efficient tracking dye in the 2-DE buffers may enlarge protein recovery on proteome maps.

Continuous marennin production by agar-entrapped Haslea ostrearia using a tubular photobioreactor with internal illumination.

The marine diatom Haslea ostrearia was immobilized in a tubular agar gel layer introduced into a photobioreactor of original design with internal illumination for the continuous synthesis of marennin, a blue-green pigment of biotechnological interest. Marennin was produced for a long-term period (27-43 days) and the volumetric productivity was maximum (18.7 mg day(-1) l(-1) gel) at the highest dilution rate (0.25 day(-1)) and lowest agar layer thickness (3 mm). Heterogeneous cell distribution in the agar layer revealed diffusional limitation of light and nutrients. However, the 3 mm gel thickness led to a more homogeneous cell distribution during incubation and to an increase of the whole biomass in the agar gel layer.

Protein synthesis in Escherichia coli at 4 degrees C.

Two-dimensional electrophoresis technology was used to investigate protein synthesis by the mesophilic bacterium Escherichia coli at low temperature. It was confirmed that protein synthesis in E. coli decreased strongly after a temperature downshift from 37 to 4 degrees C. After incubation for 150 min at 4 degrees C, however, the number of synthesized proteins represented 60% of the overall polypeptide number observed at 37 degrees C. Furthermore, the analysis of autoradiograms revealed the overexpression of 69 proteins by shocked bacteria, showing that the number of cold-induced proteins has been significantly underestimated so far.

Protein patterns of gel-entrapped Escherichia coli cells differ from those of free-floating organisms.

The two-dimensional electrophoretic patterns of cellular proteins from gel-entrapped Escherichia coli cells were compared to those of exponential- and stationary-phase free-floating organisms. The amounts of several proteins in immobilized cells were significantly different from those in free bacteria. Immobilized organisms rapidly produced a high level of dipeptide permease and a single-strand binding protein, and progressively accumulated an aldehyde dehydrogenase. Immobilization also induced a decrease in the levels of two proteins, i.e., the YFID protein and a DNA-binding, stationary-phase protein. The possible role of these proteins in the high resistance of immobilized bacteria to stresses is discussed.

Viscosity properties of mineral paraffinic base oils as a key factor in their primary biodegradability.

The primary biodegradability of two types of paraffinic base oils (solvent and catalytically dewaxed oils) and their blends was evaluated using the CEC L-33-A-93 test. The biodegradability values varied between 10% and 75%. Base oil mixtures displayed varying contents in aromatic and polar compounds and a wide range of kinematic viscosity (KV) values, from roughly 10 to 600 cSt (at 40 degrees C), while their viscosity indices were almost constant (90-100). The biodegradability of oils was closely related to their content in polycyclic aromatic hydrocarbons and was also decreasing with kinematic viscosity. For the two types of base oils, a linear relationship could be set between the biodegradation percentages and the logarithms of KV values. These results show that, beside overall chemical features such as the contents in aromatic compounds, KV may be a prominent parameter for assessing the primary biodegradability of mineral base oils.

Continuous alcoholic fermentation of glucose/xylose mixtures by co-immobilized Saccharomyces cerevisiae and Candida shehatae.

Viable Saccharomyces cerevisiae and Candida shehatae cells were co-immobilized in a composite agar layer/microporous membrane structure. This immobilized-cell structure was placed in a vertical position between the two halves of a double-chambered, stainless-steel bioreactor of original design and applied to the continuous alcoholic fermentation of a mixture of glucose (35 g dm-3) and xylose (15 g dm-3). Various dilution rates and initial cell loadings of the gel layer were tested. Simultaneous consumption of the two sugars was always observed. The best fermentation performance was obtained at low dilution rate (0.02 h-1) with an excess of C. shehatae over S. cerevisiae in the initial cell loading of the gel (5.0 mg dry weight and 0.65 mg dry weight cm-3 gel respectively): 100% of glucose and 73% of xylose were consumed with an ethanol yield coefficient of 0.48 g g total sugars-1. In these conditions, however, the ethanol production rate per unit volume of gel remained low (0.37 g h-1 dm-3). Viable cell counts in gel samples after incubation highlighted significant heterogeneities in the spatial distribution of the two yeast species in both the vertical and the transverse directions. In particular, the overall cell number decreased from the bottom to the top of the agar sheet, which may explain the low ethanol productivity relative to the total gel volume.

Antibacterial activity of synthetic dermaseptins against growing and non-growing Escherichia coli cultures.

We synthesized, and purified to homogeneity, four peptides with sequences corresponding to natural dermaseptins. We compared their in-vitro antibacterial potencies against growing and non-growing Escherichia coli cells under different oxygen conditions. The dermaseptins displayed different antibacterial potencies (MICs = 1-4 microM). Killing assays showed that slowly growing bacteria were more susceptible to the synthetic replicates of dermaseptins than rapidly growing cells. In addition, the peptides displayed high efficacy in strictly anaerobic incubation conditions. These results suggest that synthetic dermaseptins are of potential interest in the eradication of bacteria placed in a dormant state and/or subjected to low oxygen tensions.

Resistance of artificial biofilms of Pseudomonas aeruginosa to imipenem and tobramycin.

Viable cells of Pseudomonas aeruginosa were entrapped in alginate gel layers and incubated in a minimal glucose (15 g/L)-yeast extract (2 g/L)-salt medium to form artificial biofilm-like structures. After cultivation for 2 days, the biomass distribution inside the polymer was highly heterogeneous. The cell number reached approximately 1011 cells/g gel in the outer regions of the gel structures whereas the inner areas were less colonized (c. 10(8) cells g/gel). Killing of immobilized organisms by imipenem and tobramycin were compared with free-cell experiments (inoculum c. 10(9) cells/mL). Sessile-like bacteria displayed a higher resistance to the two antibiotics used alone or in combination than did suspended cells. Exposure for 10 h to 20 x MIC imipenem and 15 x MIC tobramycin reduced the number of viable immobilized bacteria to 0.3% and 3%, respectively, of the initial cell population, whereas these antibiotic concentrations were much more efficient (bactericidal) against free-cell cultures (5 log kill in 6 h). A synergic effect of tobramycin and imipenem was detected on bacterial suspensions but not on biofilm-like structures. Effective diffusivity measurements showed that the diffusion of imipenem in the alginate layer was not hindered. A slight but significant enhancement of beta-lactamase induction in immobilized cells as compared with their suspended counterparts was insufficient to explain the high resistance of sessile-like bacteria.

Antibacterial efficacy of tobramycin against anaerobic Escherichia coli cultures in the presence of electron acceptors.

The antimicrobial activity of tobramycin against anaerobic cultures of Escherichia coli was tested in the presence of various electron carriers. The presence of 2,6-dichlorophenol 4-indophenol (DCIP) significantly enhanced the killing efficacy of tobramycin. Only 0.003% of the initial cell population (i.e. 10(6) cfu/mL) remained viable after exposure for 10 h to the mixture of antibiotic (20 x MIC, i.e. 40 mg/L) and electron acceptor (10(-3) M), as compared with 9% of surviving organisms in the presence of tobramycin alone. Less synergy was obtained with p-benzoquinone and 1,2-naphthoquinone. Fumarate did not affect the efficiency of the antibiotic. The mixture of tobramycin and DCIP was ineffective against agar-entrapped bacteria which, like biofilm organisms, are subject to oxygen limitation.

Underexpression of porin protein OmpF in agar-entrapped, sessile-like Escherichia coli.

The electrophoretic patterns of the outer membrane proteins of agar-entrapped Escherichia coli cells were found to be different from those of free organisms. In particular, the porin protein OmpF was underexpressed in immobilized bacteria, that displayed enhanced resistance to latamoxef.

The role of oxygen limitation in the resistance of agar-entrapped, sessile-like Escherichia coli to aminoglycoside and beta-lactam antibiotics.

Viable cells of Escherichia coli were entrapped in agar gel layers to form artificial biofilm-like structures. Killing assays of immobilized organisms by latamoxef and tobramycin were performed under different oxygenation conditions of the culture medium and compared with free-cell experiments. Under moderate aeration, agar-entrapped bacteria displayed higher resistance to the two antibiotics than suspended cells. Slow-growing free-cell cultures were resistant to latamoxef but not to tobramycin. In anaerobic incubation conditions, suspended organisms were highly resistant to the two antibiotics. Sustained oxygenation enhanced tobramycin efficacy against free and immobilized cells. These results show that oxygen deficiency in the gel layer contributes to the enhanced antibiotic resistance of sessile-like cells.

Susceptibility of agar-entrapped Escherichia coli cultures to cefotaxime as assessed by the potentiometric measurement of lipoic acid reduction.

We used an original potentiometric procedure to monitor the metabolic activity of biofilm-like structures and to assess their susceptibility to betalactam antibiotics. The reduction of exogeneous alpha-lipoic acid to dihydrolipoic acid by planktonic and agar-entrapped bacteria, metabolizing glucose, was followed by potential-time measurements using gold versus reference electrodes. Immobilized cultures displayed a lower susceptibility to cefotaxime than their free counterparts. The redox potential-time courses showed that the gel matrix was more rapidly oxygen depleted than the free-cell culture medium. The depletion of both glucose and dissolved O2 inside the gel induced a breakdown in the reducing activity of agar-entrapped organisms.